cre recombinase  (New England Biolabs)


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    Structured Review

    New England Biolabs cre recombinase
    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre <t>recombinase</t> in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
    Cre Recombinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum"

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00273

    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
    Figure Legend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Techniques Used: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

    2) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    3) Product Images from "Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum"

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00273

    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
    Figure Legend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Techniques Used: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

    4) Product Images from "A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid"

    Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0462-x

    Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation
    Figure Legend Snippet: Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation

    Techniques Used: Plasmid Preparation, Incubation, Transformation Assay

    Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion
    Figure Legend Snippet: Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion

    Techniques Used: Plasmid Preparation, In Vitro

    5) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.
    Figure Legend Snippet: Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Labeling, Autoradiography

    6) Product Images from "Modular assembly of transposon integratable multigene vectors using RecWay assembly"

    Article Title: Modular assembly of transposon integratable multigene vectors using RecWay assembly

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt115

    Assembly of up to 6 gene vectors using Cre Recombinase. ( a ) Diagram of the two dual DEST Shuttle vectors used to subsequently generate four or six gene vectors after dual LR Clonase reaction. The Cre recombinase LoxP and Lox2722 mutant are represented as black and white triangles, respectively. ( b ) Diagram of vector layout of the insulated versions of the shuttle vectors and final PB dual DEST vectors are also shown. ( c ) Schematic of steps to generate bacterial marker-free six gene transposon vectors using RecWay assembly. Three dual clonase reactions are performed using two shuttle vectors and a single transposon dual DEST vector. I-SceI digestion and self-ligation of shuttle vectors containing the cDNAs/shRNAs of interest is performed followed by retrofitting via Cre recombination of all three dual vectors. Once assembled, vectors can be transformed into FLP recombinase-expressing bacteria to remove the Kan R (kanamycin), Cm R (chloramphenicol) and Spec R (spectinomycin) bacterial markers flanked by unique FRT sites (FRT1, 3 and 14, respectively). The FRT sites and insulators are not shown for simplicity; see Supplementary Figure S1a and b for a more highly detailed schematic representation. ( d ) Seven-day timeline for the assembly of bacterial marker-free six gene transposon vectors using RecWay assembly.
    Figure Legend Snippet: Assembly of up to 6 gene vectors using Cre Recombinase. ( a ) Diagram of the two dual DEST Shuttle vectors used to subsequently generate four or six gene vectors after dual LR Clonase reaction. The Cre recombinase LoxP and Lox2722 mutant are represented as black and white triangles, respectively. ( b ) Diagram of vector layout of the insulated versions of the shuttle vectors and final PB dual DEST vectors are also shown. ( c ) Schematic of steps to generate bacterial marker-free six gene transposon vectors using RecWay assembly. Three dual clonase reactions are performed using two shuttle vectors and a single transposon dual DEST vector. I-SceI digestion and self-ligation of shuttle vectors containing the cDNAs/shRNAs of interest is performed followed by retrofitting via Cre recombination of all three dual vectors. Once assembled, vectors can be transformed into FLP recombinase-expressing bacteria to remove the Kan R (kanamycin), Cm R (chloramphenicol) and Spec R (spectinomycin) bacterial markers flanked by unique FRT sites (FRT1, 3 and 14, respectively). The FRT sites and insulators are not shown for simplicity; see Supplementary Figure S1a and b for a more highly detailed schematic representation. ( d ) Seven-day timeline for the assembly of bacterial marker-free six gene transposon vectors using RecWay assembly.

    Techniques Used: Mutagenesis, Plasmid Preparation, Marker, Ligation, Transformation Assay, Expressing

    7) Product Images from "Digital switching in a biosensor circuit via programmable timing of gene availability"

    Article Title: Digital switching in a biosensor circuit via programmable timing of gene availability

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1680

    Control of the timing a-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin ( Supplementary Fig. 4 ). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.
    Figure Legend Snippet: Control of the timing a-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin ( Supplementary Fig. 4 ). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.

    Techniques Used: Expressing, Plasmid Preparation, Activity Assay, Concentration Assay, Translocation Assay, Transfection, Flow Cytometry, Cytometry, Standard Deviation

    8) Product Images from "Digital switching in a biosensor circuit via programmable timing of gene availability"

    Article Title: Digital switching in a biosensor circuit via programmable timing of gene availability

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1680

    Control of the timing a-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin ( Supplementary Fig. 4 ). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.
    Figure Legend Snippet: Control of the timing a-c The top, the middle and the bottom charts rows correspond to On state, Off state and On:Off ratio, respectively. The On:Off curves are calculated by dividing the fitted curves of the On by the Off measurements. a Delayed sensor characterization as a function of Cre-expressing plasmid amount. The curves are fitted to Hill function with n = 1 (On states) and to power law (Off states). b Modulation of Cre expression with an inducible promoter. ETR-iCre activity was ascertained from ETR-DsRed reporter with various concentration of erythromycin ( Supplementary Fig. 4 ). X-axis shows percentage of promoter activity relative to full activity in absence of Erythromycin. The curves are fitted to linear (On states) and exponential (Off states) regressions. c Chemical delay of ER2-Cre-ER2 translocation. X-axis shows the time (hours) between the transfection and the addition of Tamoxifen. The curves are fitted to linear (On states) and exponential (Off states) regressions. d Recombinase cascade eliminates the leakage. Sensor diagram is shown (left). The bar chart compares standard architecture with a Cre-only delayed architecture to a cascade system, with the representative flow cytometry scatter plots shown on the right. Error bars show ± standard deviation from at least three biological replicas.

    Techniques Used: Expressing, Plasmid Preparation, Activity Assay, Concentration Assay, Translocation Assay, Transfection, Flow Cytometry, Cytometry, Standard Deviation

    9) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.
    Figure Legend Snippet: Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Labeling, Autoradiography

    10) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.
    Figure Legend Snippet: Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Labeling, Autoradiography

    11) Product Images from "A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing"

    Article Title: A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep37563

    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.
    Figure Legend Snippet: Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.

    Techniques Used: Sequencing, Ligation, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Mutagenesis, Binding Assay, Generated

    Development and characterization of an optimized emulsion PCR protocol. (a) An optimized, large scale, emulsion PCR protocol was developed to reduce template switching in the generation of amplicons for Ion Torrent and Illumina sequencing. A 50 μl PCR reaction (Phusion Hot Start with green buffer) was mixed with mineral oil and surfactant. (b) The mix was converted into a large scale emulsion using a Fast Prep homogenizer in 5 minutes. (c) To evaluate the stability and size distribution of the formed micelles, three separate PCR reactions were prepared and labels with different water soluble fluorophores (DyLight 488, 549 and 650 respectively) and made into three emulsion reactions. After mixing together the three emulsions through repeated pipetting the reaction was imaged and quantified using laser-scanning confocal microscopy emulsion. Quantification resulted in a mean diameter of the micelles of 3.7 ± 2.3 μm and a total micelle count of 2.6 × 10 8 per individual reaction. (d) The emulsion was then divided into 6 individual PCR tubes and covered with mineral oil, which remain stable after PCR. (e) Isobutanol was then used to break the emulsion. (f) The compartmentalization of a PCR reaction by emPCR enables even amplification of an equimolar mixture of two oligonucleotides (126 and 150 bp long) with identical flanking sequences but with known difference in PCR efficiency. (g) Formation of chimeras due to template switching was analysed through a comparative experiment utilizing the long amplicon library 3, containing a 1 kb long stretch of constitutive backbone which can be removed using Cre-recombinase ( Fig. 3c ). With regular PCR, this library displayed extensive template switching regardless of PCR protocol or length of constitutive sequence. (h) Using emPCR on the other hand, the template switching could be significantly reduced to a level where it became insensitive to both changes in PCR protocol and constitutive sequence length.
    Figure Legend Snippet: Development and characterization of an optimized emulsion PCR protocol. (a) An optimized, large scale, emulsion PCR protocol was developed to reduce template switching in the generation of amplicons for Ion Torrent and Illumina sequencing. A 50 μl PCR reaction (Phusion Hot Start with green buffer) was mixed with mineral oil and surfactant. (b) The mix was converted into a large scale emulsion using a Fast Prep homogenizer in 5 minutes. (c) To evaluate the stability and size distribution of the formed micelles, three separate PCR reactions were prepared and labels with different water soluble fluorophores (DyLight 488, 549 and 650 respectively) and made into three emulsion reactions. After mixing together the three emulsions through repeated pipetting the reaction was imaged and quantified using laser-scanning confocal microscopy emulsion. Quantification resulted in a mean diameter of the micelles of 3.7 ± 2.3 μm and a total micelle count of 2.6 × 10 8 per individual reaction. (d) The emulsion was then divided into 6 individual PCR tubes and covered with mineral oil, which remain stable after PCR. (e) Isobutanol was then used to break the emulsion. (f) The compartmentalization of a PCR reaction by emPCR enables even amplification of an equimolar mixture of two oligonucleotides (126 and 150 bp long) with identical flanking sequences but with known difference in PCR efficiency. (g) Formation of chimeras due to template switching was analysed through a comparative experiment utilizing the long amplicon library 3, containing a 1 kb long stretch of constitutive backbone which can be removed using Cre-recombinase ( Fig. 3c ). With regular PCR, this library displayed extensive template switching regardless of PCR protocol or length of constitutive sequence. (h) Using emPCR on the other hand, the template switching could be significantly reduced to a level where it became insensitive to both changes in PCR protocol and constitutive sequence length.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Confocal Microscopy, Amplification

    12) Product Images from "Modular assembly of transposon integratable multigene vectors using RecWay assembly"

    Article Title: Modular assembly of transposon integratable multigene vectors using RecWay assembly

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt115

    Assembly of up to 6 gene vectors using Cre Recombinase. ( a ) Diagram of the two dual DEST Shuttle vectors used to subsequently generate four or six gene vectors after dual LR Clonase reaction. The Cre recombinase LoxP and Lox2722 mutant are represented as black and white triangles, respectively. ( b ) Diagram of vector layout of the insulated versions of the shuttle vectors and final PB dual DEST vectors are also shown. ( c ) Schematic of steps to generate bacterial marker-free six gene transposon vectors using RecWay assembly. Three dual clonase reactions are performed using two shuttle vectors and a single transposon dual DEST vector. I-SceI digestion and self-ligation of shuttle vectors containing the cDNAs/shRNAs of interest is performed followed by retrofitting via Cre recombination of all three dual vectors. Once assembled, vectors can be transformed into FLP recombinase-expressing bacteria to remove the Kan R (kanamycin), Cm R (chloramphenicol) and Spec R (spectinomycin) bacterial markers flanked by unique FRT sites (FRT1, 3 and 14, respectively). The FRT sites and insulators are not shown for simplicity; see Supplementary Figure S1a and b for a more highly detailed schematic representation. ( d ) Seven-day timeline for the assembly of bacterial marker-free six gene transposon vectors using RecWay assembly.
    Figure Legend Snippet: Assembly of up to 6 gene vectors using Cre Recombinase. ( a ) Diagram of the two dual DEST Shuttle vectors used to subsequently generate four or six gene vectors after dual LR Clonase reaction. The Cre recombinase LoxP and Lox2722 mutant are represented as black and white triangles, respectively. ( b ) Diagram of vector layout of the insulated versions of the shuttle vectors and final PB dual DEST vectors are also shown. ( c ) Schematic of steps to generate bacterial marker-free six gene transposon vectors using RecWay assembly. Three dual clonase reactions are performed using two shuttle vectors and a single transposon dual DEST vector. I-SceI digestion and self-ligation of shuttle vectors containing the cDNAs/shRNAs of interest is performed followed by retrofitting via Cre recombination of all three dual vectors. Once assembled, vectors can be transformed into FLP recombinase-expressing bacteria to remove the Kan R (kanamycin), Cm R (chloramphenicol) and Spec R (spectinomycin) bacterial markers flanked by unique FRT sites (FRT1, 3 and 14, respectively). The FRT sites and insulators are not shown for simplicity; see Supplementary Figure S1a and b for a more highly detailed schematic representation. ( d ) Seven-day timeline for the assembly of bacterial marker-free six gene transposon vectors using RecWay assembly.

    Techniques Used: Mutagenesis, Plasmid Preparation, Marker, Ligation, Transformation Assay, Expressing

    13) Product Images from "Polylox barcoding reveals haematopoietic stem cell fates realized in vivo"

    Article Title: Polylox barcoding reveals haematopoietic stem cell fates realized in vivo

    Journal: Nature

    doi: 10.1038/nature23653

    Polylox : A Cre recombinase-driven artificial DNA recombination substrate. a , Structure of the Polylox cassette with loxP sites (triangles; black and white split symbolizes recombination site). Colored linkers represent DNA segments ‘1’-‘9’. Examples for recombination products resulting from one Cre-mediated excision, and one Cre-mediated inversion are shown. The original code segments (‘letters’) are abbreviated ‘1’-‘9’, and their inversions ‘A’-‘I’. b , In vitro digestion of Polylox DNA insert in pWP-AG vector by Cre recombinase, and size resolution of recombination products by gel electrophoresis ( Supplementary Fig. 1a ).
    Figure Legend Snippet: Polylox : A Cre recombinase-driven artificial DNA recombination substrate. a , Structure of the Polylox cassette with loxP sites (triangles; black and white split symbolizes recombination site). Colored linkers represent DNA segments ‘1’-‘9’. Examples for recombination products resulting from one Cre-mediated excision, and one Cre-mediated inversion are shown. The original code segments (‘letters’) are abbreviated ‘1’-‘9’, and their inversions ‘A’-‘I’. b , In vitro digestion of Polylox DNA insert in pWP-AG vector by Cre recombinase, and size resolution of recombination products by gel electrophoresis ( Supplementary Fig. 1a ).

    Techniques Used: In Vitro, Plasmid Preparation, Nucleic Acid Electrophoresis

    14) Product Images from "Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction"

    Article Title: Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction

    Journal: Life

    doi: 10.3390/life8040043

    Effect of Cre- loxP system on the concatemer production in RCR. pUC_OriC300 (− loxP ) or pUC_OLDT (+ loxP ) (0.05 ng) was incubated in the RCR mixture at 33 °C for 3 h in the absence (0 mU) or presence (5, 15, 50, or 150 mU) of Cre recombinase. The product was analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining. The ratio of concatemers to the sum of concatemers and supercoils is shown in a graph.
    Figure Legend Snippet: Effect of Cre- loxP system on the concatemer production in RCR. pUC_OriC300 (− loxP ) or pUC_OLDT (+ loxP ) (0.05 ng) was incubated in the RCR mixture at 33 °C for 3 h in the absence (0 mU) or presence (5, 15, 50, or 150 mU) of Cre recombinase. The product was analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining. The ratio of concatemers to the sum of concatemers and supercoils is shown in a graph.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    15) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    16) Product Images from "A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid"

    Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0462-x

    Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation
    Figure Legend Snippet: Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation

    Techniques Used: Plasmid Preparation, Incubation, Transformation Assay

    Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion
    Figure Legend Snippet: Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion

    Techniques Used: Plasmid Preparation, In Vitro

    17) Product Images from "Taok2 Controls Behavioral Response to Ethanol in Mice"

    Article Title: Taok2 Controls Behavioral Response to Ethanol in Mice

    Journal: Genes, brain, and behavior

    doi: 10.1111/j.1601-183X.2012.00834.x

    Targeted disruption of the Taok2 gene in mice (a) Schematic of the conditionally targeted Taok2 allele. The Taok2tm1 allele was generated by introduction of lox P sites flanking exons 2 though 7 of the Taok2 gene. Within intron 7 is a neomycin resistance gene flanked by lox P and frt sites. Upon crossing this line to mice expressing Flpe recombinase, the neomycin selectable marker is removed along with one of the lox P sites to generate the Taok2tm1fl allele. In the presence of Cre recombinase, the remaining lox P sites are recombined into a single lox P site, removing exons 2 though 7 and the translational start site to generate the Taok2tm1Δ allele. (b) Brain sagittal section of a control mouse shows strong expression of TAOK2 by immunohistological analysis in all areas, especially in cortex, hippocampus and striatum. (c) Immunohistological analysis shows reduced TAOK2 expression in Taok2tm1fl/fl;Nes-cre brain. (d) Quantitative PCR analysis of total mouse brain extracts shows near absence of Taok2 transcript in Taok2tm1fl/fl;Nes-cre brain ( n = 4) compared to control mice ( n = 2). (e) Western blot showing comparative loss of TAOK2 protein in total brain lysates of Taok2tm1fl/fl;Nes-cre mice relative to controls. (f) Quantification of anti-TAOK2 signal from (e) comparing Taok2tm1fl/fl;Nes-cre mice ( n = 2) and controls ( n = 2). Lox P sites are denoted by grey chevrons, frt sites by black chevrons. Neo = neomycin resistance gene. The positions of restriction sites BamH1 and BsrB1 used in the generation of the targeting construct are indicated. CTX = cortex; HC = hippocampus; STR = striatum. Error bars are mean ± SEM. Asterisks indicate level of significance (* P
    Figure Legend Snippet: Targeted disruption of the Taok2 gene in mice (a) Schematic of the conditionally targeted Taok2 allele. The Taok2tm1 allele was generated by introduction of lox P sites flanking exons 2 though 7 of the Taok2 gene. Within intron 7 is a neomycin resistance gene flanked by lox P and frt sites. Upon crossing this line to mice expressing Flpe recombinase, the neomycin selectable marker is removed along with one of the lox P sites to generate the Taok2tm1fl allele. In the presence of Cre recombinase, the remaining lox P sites are recombined into a single lox P site, removing exons 2 though 7 and the translational start site to generate the Taok2tm1Δ allele. (b) Brain sagittal section of a control mouse shows strong expression of TAOK2 by immunohistological analysis in all areas, especially in cortex, hippocampus and striatum. (c) Immunohistological analysis shows reduced TAOK2 expression in Taok2tm1fl/fl;Nes-cre brain. (d) Quantitative PCR analysis of total mouse brain extracts shows near absence of Taok2 transcript in Taok2tm1fl/fl;Nes-cre brain ( n = 4) compared to control mice ( n = 2). (e) Western blot showing comparative loss of TAOK2 protein in total brain lysates of Taok2tm1fl/fl;Nes-cre mice relative to controls. (f) Quantification of anti-TAOK2 signal from (e) comparing Taok2tm1fl/fl;Nes-cre mice ( n = 2) and controls ( n = 2). Lox P sites are denoted by grey chevrons, frt sites by black chevrons. Neo = neomycin resistance gene. The positions of restriction sites BamH1 and BsrB1 used in the generation of the targeting construct are indicated. CTX = cortex; HC = hippocampus; STR = striatum. Error bars are mean ± SEM. Asterisks indicate level of significance (* P

    Techniques Used: Mouse Assay, Generated, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Construct

    18) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.
    Figure Legend Snippet: Electrophoresis of radiolabeled transcription and translation-coupled DNA replication (TTcDR) products. The TTcDR reaction was performed with [ 32 P]-dCTP with or without 30 mU/μl Cre recombinase at 30 °C for 16 h. Before and after the degradation of linear DNAs with an exonuclease, the product DNA was subjected to 1% agarose gel electrophoresis, and [ 32 P]-labeled DNA was detected by autoradiography. ( a ) Autoradiography of the TTcDR products of clone 6-wt-loxP (0.40 nM) with or without 30 mU/μl Cre recombinase before and after exonuclease treatment. Since the amount of DNA was too large before exonuclease treatment, we also applied 1/10 volume of the samples. The control circular DNA was applied in lane C. A lower contrast image is shown in Fig. S5 . ( b ) Autoradiography of the TTcDR products of the original DNA, clone 6, and clone 6-wt-loxP. TTcDR reactions were performed in the presence of 30 mU/μl Cre recombinase and subjected to electrophoresis after exonuclease treatment.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Labeling, Autoradiography

    19) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    20) Product Images from "A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid"

    Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0462-x

    Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation
    Figure Legend Snippet: Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation

    Techniques Used: Plasmid Preparation, Incubation, Transformation Assay

    Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion
    Figure Legend Snippet: Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion

    Techniques Used: Plasmid Preparation, In Vitro

    21) Product Images from "Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination"

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31585-1

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).
    Figure Legend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).
    Figure Legend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.
    Figure Legend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
    Figure Legend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Techniques Used: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.
    Figure Legend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Techniques Used: Sequencing

    22) Product Images from "Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum"

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00273

    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
    Figure Legend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Techniques Used: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

    23) Product Images from "Taok2 Controls Behavioral Response to Ethanol in Mice"

    Article Title: Taok2 Controls Behavioral Response to Ethanol in Mice

    Journal: Genes, brain, and behavior

    doi: 10.1111/j.1601-183X.2012.00834.x

    Targeted disruption of the Taok2 gene in mice (a) Schematic of the conditionally targeted Taok2 allele. The Taok2tm1 allele was generated by introduction of lox P sites flanking exons 2 though 7 of the Taok2 gene. Within intron 7 is a neomycin resistance gene flanked by lox P and frt sites. Upon crossing this line to mice expressing Flpe recombinase, the neomycin selectable marker is removed along with one of the lox P sites to generate the Taok2tm1fl allele. In the presence of Cre recombinase, the remaining lox P sites are recombined into a single lox P site, removing exons 2 though 7 and the translational start site to generate the Taok2tm1Δ allele. (b) Brain sagittal section of a control mouse shows strong expression of TAOK2 by immunohistological analysis in all areas, especially in cortex, hippocampus and striatum. (c) Immunohistological analysis shows reduced TAOK2 expression in Taok2tm1fl/fl;Nes-cre brain. (d) Quantitative PCR analysis of total mouse brain extracts shows near absence of Taok2 transcript in Taok2tm1fl/fl;Nes-cre brain ( n = 4) compared to control mice ( n = 2). (e) Western blot showing comparative loss of TAOK2 protein in total brain lysates of Taok2tm1fl/fl;Nes-cre mice relative to controls. (f) Quantification of anti-TAOK2 signal from (e) comparing Taok2tm1fl/fl;Nes-cre mice ( n = 2) and controls ( n = 2). Lox P sites are denoted by grey chevrons, frt sites by black chevrons. Neo = neomycin resistance gene. The positions of restriction sites BamH1 and BsrB1 used in the generation of the targeting construct are indicated. CTX = cortex; HC = hippocampus; STR = striatum. Error bars are mean ± SEM. Asterisks indicate level of significance (* P
    Figure Legend Snippet: Targeted disruption of the Taok2 gene in mice (a) Schematic of the conditionally targeted Taok2 allele. The Taok2tm1 allele was generated by introduction of lox P sites flanking exons 2 though 7 of the Taok2 gene. Within intron 7 is a neomycin resistance gene flanked by lox P and frt sites. Upon crossing this line to mice expressing Flpe recombinase, the neomycin selectable marker is removed along with one of the lox P sites to generate the Taok2tm1fl allele. In the presence of Cre recombinase, the remaining lox P sites are recombined into a single lox P site, removing exons 2 though 7 and the translational start site to generate the Taok2tm1Δ allele. (b) Brain sagittal section of a control mouse shows strong expression of TAOK2 by immunohistological analysis in all areas, especially in cortex, hippocampus and striatum. (c) Immunohistological analysis shows reduced TAOK2 expression in Taok2tm1fl/fl;Nes-cre brain. (d) Quantitative PCR analysis of total mouse brain extracts shows near absence of Taok2 transcript in Taok2tm1fl/fl;Nes-cre brain ( n = 4) compared to control mice ( n = 2). (e) Western blot showing comparative loss of TAOK2 protein in total brain lysates of Taok2tm1fl/fl;Nes-cre mice relative to controls. (f) Quantification of anti-TAOK2 signal from (e) comparing Taok2tm1fl/fl;Nes-cre mice ( n = 2) and controls ( n = 2). Lox P sites are denoted by grey chevrons, frt sites by black chevrons. Neo = neomycin resistance gene. The positions of restriction sites BamH1 and BsrB1 used in the generation of the targeting construct are indicated. CTX = cortex; HC = hippocampus; STR = striatum. Error bars are mean ± SEM. Asterisks indicate level of significance (* P

    Techniques Used: Mouse Assay, Generated, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Construct

    24) Product Images from "Barcoded Rational AAV Vector Evolution enables systematic in vivo mapping of peptide binding motifs"

    Article Title: Barcoded Rational AAV Vector Evolution enables systematic in vivo mapping of peptide binding motifs

    Journal: bioRxiv

    doi: 10.1101/335372

    Assessment of retrograde transport in the brain using the BRAVE AAV capsid library approach (A) Pie-chart displaying the selected 131 proteins with documented affinity to synapses. (B) Schematics of BRAVE procedure; 1. NCBI reference amino acid (aa) sequences were computationally digested into 14aa (or 22aa) long polypeptides with a 1aa shifting sliding window. 2. Three alternative linkers were added to the 14aa polypeptides e.g., a rigid linker with 5 Alanine residues (14aaA5). 3. 92,358 codon optimized sequences were synthesized in parallel on an oligonucleotide array. 4. The pool of oligonucleotides was assembled into a novel AAV production backbone with Cis- acting AAV2 Rep/Cap and ITR-flanking CMV-GFP. A 20bp random molecular barcode (BC) was simultaneously inserted in the 3’ UTR of the GFP gene. 5a. Using a fraction of the plasmid prep, exposed to Cre-recombinase in vitro , a look-up table (LUT) was generated linking BC to peptide. 5b. Using the same plasmid pool from step 4, an AAV was produced (MNMlib) and multiple parallel screening experiments were performed both in vitro and in vivo followed by BCs sequencing from mRNA 6. Through the combination of the sequenced barcodes and the LUT, efficacy can be mapped back to the original 131 proteins and consensus motifs can be determined using the Hammock, hidden Markov-model based clustering approach ( 7. ). ( C-C’’’ ) First proof-of-concept study using BRAVE to re-introduce tropism for HEK293T cells in vitro . Wild-type AAV2 displays very high infectivity attributed to Heparin Sulfate (HS) proteoglycan binding ( C ). The MNMnull serotype disrupts this binding ( C’ ). The first novel capsid generated from single-generation BRAVE screening in HEK293T, MNM001, displayed a significantly recovered tropism ( C’’ ). ( D-D’’’ ) In a second experiment, we used the BRAVE technology to improve the infectivity of primary cortical rat neurons in vitro . Both AAV2-WT and the MNMnull vector displays very poor infectivity of primary neurons ( D-D’ ) and the MNM001 displays some improvement ( D’’ ). BRAVE screening in primary neurons identified a number of peptides clustering over a C-terminal region of the HSV-2 pUL1 protein which improved the infectivity of primary neurons in culture dramatically ( D’’’ ). ( E ) In vivo expression pattern of GFP after injection of 30cpc library into the rat forebrain compared to an AAV2-WT vector at the same titer.( F ) Connectivity diagram showing the injection site (striatum, Str) and connected neuronal populations frontal cortex (PFC), thalamus (Thal) and substantia nigra (SNpc) utilized for BRAVE screening in vivo for retrograde transport. ( G ) Polar plot showing the absolute quantities of the unique peptides recovered at each step of the BRAVE assay. ( H ) From the in vivo screening for improved retrograde transport capacity, we selected 23 peptides from 20 proteins that all were represented by multiple barcodes and found in multiple animals (See Supplementary Document S14). 23 of the 25 de novo AAV capsid structures allowed for higher than or at par with AAV2-WT packaging efficacy ( Supplementary Fig. S1 ), all with retrograde transport ability ( Supplementary Fig. S7 ).Davidsson et al., 2018. In vivo single-generation AAV-screen
    Figure Legend Snippet: Assessment of retrograde transport in the brain using the BRAVE AAV capsid library approach (A) Pie-chart displaying the selected 131 proteins with documented affinity to synapses. (B) Schematics of BRAVE procedure; 1. NCBI reference amino acid (aa) sequences were computationally digested into 14aa (or 22aa) long polypeptides with a 1aa shifting sliding window. 2. Three alternative linkers were added to the 14aa polypeptides e.g., a rigid linker with 5 Alanine residues (14aaA5). 3. 92,358 codon optimized sequences were synthesized in parallel on an oligonucleotide array. 4. The pool of oligonucleotides was assembled into a novel AAV production backbone with Cis- acting AAV2 Rep/Cap and ITR-flanking CMV-GFP. A 20bp random molecular barcode (BC) was simultaneously inserted in the 3’ UTR of the GFP gene. 5a. Using a fraction of the plasmid prep, exposed to Cre-recombinase in vitro , a look-up table (LUT) was generated linking BC to peptide. 5b. Using the same plasmid pool from step 4, an AAV was produced (MNMlib) and multiple parallel screening experiments were performed both in vitro and in vivo followed by BCs sequencing from mRNA 6. Through the combination of the sequenced barcodes and the LUT, efficacy can be mapped back to the original 131 proteins and consensus motifs can be determined using the Hammock, hidden Markov-model based clustering approach ( 7. ). ( C-C’’’ ) First proof-of-concept study using BRAVE to re-introduce tropism for HEK293T cells in vitro . Wild-type AAV2 displays very high infectivity attributed to Heparin Sulfate (HS) proteoglycan binding ( C ). The MNMnull serotype disrupts this binding ( C’ ). The first novel capsid generated from single-generation BRAVE screening in HEK293T, MNM001, displayed a significantly recovered tropism ( C’’ ). ( D-D’’’ ) In a second experiment, we used the BRAVE technology to improve the infectivity of primary cortical rat neurons in vitro . Both AAV2-WT and the MNMnull vector displays very poor infectivity of primary neurons ( D-D’ ) and the MNM001 displays some improvement ( D’’ ). BRAVE screening in primary neurons identified a number of peptides clustering over a C-terminal region of the HSV-2 pUL1 protein which improved the infectivity of primary neurons in culture dramatically ( D’’’ ). ( E ) In vivo expression pattern of GFP after injection of 30cpc library into the rat forebrain compared to an AAV2-WT vector at the same titer.( F ) Connectivity diagram showing the injection site (striatum, Str) and connected neuronal populations frontal cortex (PFC), thalamus (Thal) and substantia nigra (SNpc) utilized for BRAVE screening in vivo for retrograde transport. ( G ) Polar plot showing the absolute quantities of the unique peptides recovered at each step of the BRAVE assay. ( H ) From the in vivo screening for improved retrograde transport capacity, we selected 23 peptides from 20 proteins that all were represented by multiple barcodes and found in multiple animals (See Supplementary Document S14). 23 of the 25 de novo AAV capsid structures allowed for higher than or at par with AAV2-WT packaging efficacy ( Supplementary Fig. S1 ), all with retrograde transport ability ( Supplementary Fig. S7 ).Davidsson et al., 2018. In vivo single-generation AAV-screen

    Techniques Used: Synthesized, Plasmid Preparation, In Vitro, Generated, Produced, In Vivo, Sequencing, Introduce, Infection, Binding Assay, Expressing, Injection

    Conditioned place preference in animals with selective amygdo-striatal DREADD activation We injected the MNM004 vector expressing Cre-recombinase in the dorsal striatum and a Cre-inducible (DIO) chemogenetic (DREADD) vector into the basolateral amygdala (BLA) bilaterally into wild-type rats. In the control animals, the Cre-recombinase in the MNM004 vector was replaced with GFP. The conditioned place preference test was used as place aversion test to study the effect of amygdo-striatal activation using chemogenetics.( A ) The movements patterns of the two groups did not differ significantly, with all animals preferring the corners and edges over open areas. ( B-E ) After selective induction of activity of the BLA neurons projecting to the dorsal striatum using the DREADD ligand CNO, we found a striking fear and anxiety phenotype in the animals with excessive defecation, sweating a digging and freezing (see Supplementary movie 2). This was accompanied with an increase in both ipsilateral and contralateral rotation ( B ), high speed rushes ( C ) and significantly elevated mobility ( D ). However, this did not result in any conditioning, as seen in the Preference test performed the next day ( E ). Both animals in the active group and the control group spent equal time in both chamber with no signs of conditioned place aversion.
    Figure Legend Snippet: Conditioned place preference in animals with selective amygdo-striatal DREADD activation We injected the MNM004 vector expressing Cre-recombinase in the dorsal striatum and a Cre-inducible (DIO) chemogenetic (DREADD) vector into the basolateral amygdala (BLA) bilaterally into wild-type rats. In the control animals, the Cre-recombinase in the MNM004 vector was replaced with GFP. The conditioned place preference test was used as place aversion test to study the effect of amygdo-striatal activation using chemogenetics.( A ) The movements patterns of the two groups did not differ significantly, with all animals preferring the corners and edges over open areas. ( B-E ) After selective induction of activity of the BLA neurons projecting to the dorsal striatum using the DREADD ligand CNO, we found a striking fear and anxiety phenotype in the animals with excessive defecation, sweating a digging and freezing (see Supplementary movie 2). This was accompanied with an increase in both ipsilateral and contralateral rotation ( B ), high speed rushes ( C ) and significantly elevated mobility ( D ). However, this did not result in any conditioning, as seen in the Preference test performed the next day ( E ). Both animals in the active group and the control group spent equal time in both chamber with no signs of conditioned place aversion.

    Techniques Used: Conditioned Place Preference, Activation Assay, Injection, Plasmid Preparation, Expressing, Activity Assay

    25) Product Images from "Synthetic memory circuits for programmable cell reconfiguration in plants"

    Article Title: Synthetic memory circuits for programmable cell reconfiguration in plants

    Journal: bioRxiv

    doi: 10.1101/2022.02.11.480167

    Negation (NOT) function implementation in plant cells by output gene CDS or promoter excision a , Comparison of output (Rluc) repression over time with four different negation (NOT) function designs in plant cells. Right to left, Flp targeting the output gene CDS, B3 targeting the output gene CDS, Flp targeting the output gene promoter, and B3 targeting the output gene promoter. Three separate protoplast transfections were measured, one after 24 hours, one after 48 hours and one after 64 hours. Normalised circuit output is the Rluc / Fluc luminescence (circuit output) of the NOT function plasmid, divided by the circuit output of the Constitutive Rluc construct ( Fig. 1b ) to account differences between plates and time-points (n=3-4); Crossbar, mean. Asterisks denote significance from the ANOVA’s post-hoc test Tukey’s HSD: NS = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. b , Circuit output of NOT function plasmids of the 1-input states (recombinase gene present) relative to the 0-input states (no recombinase gene present) over time. Generation 1 refers to NOT function designs where the recombinase targets the output gene CDS while generation 2 refers to the NOT function designs where the recombinase targets the output gene promoter. c , The A NIMPLY B gate combines the identify (activation) function and the negation (NOT) function and was achieved in plant cells by targeting the Flp recombinase to remove the repressive OCS terminator upstream of the output (Rluc) CDS, thus activating the circuit when only Flp was expressed, and the B3 recombinase to remove the Act2 promoter upstream of the output (Rluc), thus repressing the circuit regardless of the activity of Flp. Circuit output is the Rluc luminescence over the Fluc luminescence (n=4); Crossbar, mean. Bar colours as per Fig. 2 . Bars with different letters have a significant difference calculated by a one-way ANOVA and Tukey’s HSD test (p
    Figure Legend Snippet: Negation (NOT) function implementation in plant cells by output gene CDS or promoter excision a , Comparison of output (Rluc) repression over time with four different negation (NOT) function designs in plant cells. Right to left, Flp targeting the output gene CDS, B3 targeting the output gene CDS, Flp targeting the output gene promoter, and B3 targeting the output gene promoter. Three separate protoplast transfections were measured, one after 24 hours, one after 48 hours and one after 64 hours. Normalised circuit output is the Rluc / Fluc luminescence (circuit output) of the NOT function plasmid, divided by the circuit output of the Constitutive Rluc construct ( Fig. 1b ) to account differences between plates and time-points (n=3-4); Crossbar, mean. Asterisks denote significance from the ANOVA’s post-hoc test Tukey’s HSD: NS = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. b , Circuit output of NOT function plasmids of the 1-input states (recombinase gene present) relative to the 0-input states (no recombinase gene present) over time. Generation 1 refers to NOT function designs where the recombinase targets the output gene CDS while generation 2 refers to the NOT function designs where the recombinase targets the output gene promoter. c , The A NIMPLY B gate combines the identify (activation) function and the negation (NOT) function and was achieved in plant cells by targeting the Flp recombinase to remove the repressive OCS terminator upstream of the output (Rluc) CDS, thus activating the circuit when only Flp was expressed, and the B3 recombinase to remove the Act2 promoter upstream of the output (Rluc), thus repressing the circuit regardless of the activity of Flp. Circuit output is the Rluc luminescence over the Fluc luminescence (n=4); Crossbar, mean. Bar colours as per Fig. 2 . Bars with different letters have a significant difference calculated by a one-way ANOVA and Tukey’s HSD test (p

    Techniques Used: Transfection, Plasmid Preparation, Construct, Activation Assay, Activity Assay

    Identification of a additional functional recombinases for the construction of complex gene circuits a , Schematic of the Cre/Flp-based OR gate construct design and the Cre/Flp-based OR gate that produces high circuit output activity only when Flp is present alone, 24 hours post-transfection (as in panels c and e). Circuit output activity is the Rluc / Fluc luminescence ratio; also for panel d) 24 hours after protoplast transfection (n=4); Crossbar, mean. Blue bar represent the control sample, green bars represent samples expected to be activated (turned on), gray bars represent samples expected to be repressed (turned off) and bars with different letters have a significant difference calculated by a one-way ANOVA and Tukey’s HSD test (p
    Figure Legend Snippet: Identification of a additional functional recombinases for the construction of complex gene circuits a , Schematic of the Cre/Flp-based OR gate construct design and the Cre/Flp-based OR gate that produces high circuit output activity only when Flp is present alone, 24 hours post-transfection (as in panels c and e). Circuit output activity is the Rluc / Fluc luminescence ratio; also for panel d) 24 hours after protoplast transfection (n=4); Crossbar, mean. Blue bar represent the control sample, green bars represent samples expected to be activated (turned on), gray bars represent samples expected to be repressed (turned off) and bars with different letters have a significant difference calculated by a one-way ANOVA and Tukey’s HSD test (p

    Techniques Used: Functional Assay, Construct, Activity Assay, Transfection

    Construction of AND and NAND gates using split-recombinases a , Flp split into two recombinase fragments (F1 and F2), each fused to the phage C1 dimerisation domain, to construct an AND gate through removal of an FRT-flanked OCS terminator located in the 5’UTR of the Rluc output gene, measured either 24 hours or 48 hours after protoplast transfection. Schematic represents the 2-input construct design. Crossbar, mean (n=4). Bar colours and letters representing statistical differences per Fig. 2 . b , Flp split into two recombinase fragments (F1 and F2), each fused to the phage C1 dimerisation domain, to construct a NAND gate through removal of the FRT-flanked Act2 promoter upstream of the Rluc output gene, measured either 24 hours or 48 hours after protoplast transfection. Schematic represents the 2-input construct design. Crossbar, mean (n=4). Bar colours and letters representing statistical differences per Fig. 2 . Some figure components created with BioRender.
    Figure Legend Snippet: Construction of AND and NAND gates using split-recombinases a , Flp split into two recombinase fragments (F1 and F2), each fused to the phage C1 dimerisation domain, to construct an AND gate through removal of an FRT-flanked OCS terminator located in the 5’UTR of the Rluc output gene, measured either 24 hours or 48 hours after protoplast transfection. Schematic represents the 2-input construct design. Crossbar, mean (n=4). Bar colours and letters representing statistical differences per Fig. 2 . b , Flp split into two recombinase fragments (F1 and F2), each fused to the phage C1 dimerisation domain, to construct a NAND gate through removal of the FRT-flanked Act2 promoter upstream of the Rluc output gene, measured either 24 hours or 48 hours after protoplast transfection. Schematic represents the 2-input construct design. Crossbar, mean (n=4). Bar colours and letters representing statistical differences per Fig. 2 . Some figure components created with BioRender.

    Techniques Used: Construct, Transfection

    AND and OR gate construction using recombination-based gene circuits in plants a , The output and normaliser units of the OR gate construct design. b , c , A 2-input OR gate that produces high circuit output activity when one of the two inputs is present, (b) 24 or (c) 48 hours after Arabidopsis protoplast transfection. Circuit output activity is the Rluc / Fluc luminescence ratio; also for panel d) (n=4); Crossbar, mean. Bar colours and letters representing statistical differences per Fig. 2 . d , The output and normaliser units of the AND gate construct design. e , f , A 2-input AND gate that produces high Rluc luminescence relative to Fluc luminescence when both of the inputs are present, (e) 24 or (f) 48 hours after Arabidopsis protoplast transfection. Circuit output is the Rluc luminescence over the Fluc luminescence (n=4); Crossbar, mean. Bar colours as per Fig. 2 . g , AND gate functionality in vivo , driven by condition-specific promoters. Schematic of the construct at top. Confocal images from roots of induced (dexamethasone[DEX]-treated) and uninduced (DMSO solvent control) T3 transgenic plants, with optical transverse sections of roots shown. Expression of the output gene (nuclear localizedlocalised GFP) is blocked unless in a cell with both recombinases expressed, where Flp expression is induced when exposed to DEX and B3 expression is driven by the cortex-specific CO2 promoter. These plants also express nuclear localized mCherry from the 35S promoter and were stained with the cell wall stain propidium iodide (PI). The magenta (mCherry and PI) represents the infocus nuclei and cell walls, while the cyan (GFP) represents the induced nuclei in the cortex. Two representative plants per condition. The outermost cell layer is the epidermis (ep), then the cortex (c), and the layer below that is the endodermis (end). Scale bar is 20 μm. Some figure components created with BioRender.
    Figure Legend Snippet: AND and OR gate construction using recombination-based gene circuits in plants a , The output and normaliser units of the OR gate construct design. b , c , A 2-input OR gate that produces high circuit output activity when one of the two inputs is present, (b) 24 or (c) 48 hours after Arabidopsis protoplast transfection. Circuit output activity is the Rluc / Fluc luminescence ratio; also for panel d) (n=4); Crossbar, mean. Bar colours and letters representing statistical differences per Fig. 2 . d , The output and normaliser units of the AND gate construct design. e , f , A 2-input AND gate that produces high Rluc luminescence relative to Fluc luminescence when both of the inputs are present, (e) 24 or (f) 48 hours after Arabidopsis protoplast transfection. Circuit output is the Rluc luminescence over the Fluc luminescence (n=4); Crossbar, mean. Bar colours as per Fig. 2 . g , AND gate functionality in vivo , driven by condition-specific promoters. Schematic of the construct at top. Confocal images from roots of induced (dexamethasone[DEX]-treated) and uninduced (DMSO solvent control) T3 transgenic plants, with optical transverse sections of roots shown. Expression of the output gene (nuclear localizedlocalised GFP) is blocked unless in a cell with both recombinases expressed, where Flp expression is induced when exposed to DEX and B3 expression is driven by the cortex-specific CO2 promoter. These plants also express nuclear localized mCherry from the 35S promoter and were stained with the cell wall stain propidium iodide (PI). The magenta (mCherry and PI) represents the infocus nuclei and cell walls, while the cyan (GFP) represents the induced nuclei in the cortex. Two representative plants per condition. The outermost cell layer is the epidermis (ep), then the cortex (c), and the layer below that is the endodermis (end). Scale bar is 20 μm. Some figure components created with BioRender.

    Techniques Used: Construct, Activity Assay, Transfection, In Vivo, Transgenic Assay, Expressing, Staining

    26) Product Images from "Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans"

    Article Title: Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

    Journal: bioRxiv

    doi: 10.1101/2021.08.20.457072

    Overview of the strategy for Cre/ lox -mediated chromosomal integration. 1) The landing pad (LP) containing the bar marker gene flanked by loxP and lox2272-71 is integrated in the destination locus of Aspergillus nidulans genome by homologous recombination. The resultant strain A. nidulans LP is used to prepare protoplasts that can be stored for subsequent transformations. 2) A. nidulans LP protoplasts are transformed with the donor vector that contains loxP and lox2272-66 flanking the marker gene pyrG , a fluorescent reporter and the genes of interest, along with a second vector for transient expression of cre recombinase. Stable integration can be achieved by LE/RE in 1-step recombination or in 2-steps by RMCE. 3) Selection of the recombinant colonies in minimal media for pyrG complementation.
    Figure Legend Snippet: Overview of the strategy for Cre/ lox -mediated chromosomal integration. 1) The landing pad (LP) containing the bar marker gene flanked by loxP and lox2272-71 is integrated in the destination locus of Aspergillus nidulans genome by homologous recombination. The resultant strain A. nidulans LP is used to prepare protoplasts that can be stored for subsequent transformations. 2) A. nidulans LP protoplasts are transformed with the donor vector that contains loxP and lox2272-66 flanking the marker gene pyrG , a fluorescent reporter and the genes of interest, along with a second vector for transient expression of cre recombinase. Stable integration can be achieved by LE/RE in 1-step recombination or in 2-steps by RMCE. 3) Selection of the recombinant colonies in minimal media for pyrG complementation.

    Techniques Used: Marker, Homologous Recombination, Transformation Assay, Plasmid Preparation, Expressing, Selection, Recombinant

    Validation of the double mutant site lox2272-66/71 . A. Experimental set up. Fragments were amplified by PCR containing the site lox2272-66 or lox2272-72 . After in vitro recombination mediated by Cre, two recombination products are formed of different length compared to the substrates of recombination. B. Gel electrophoresis run after in vitro recombination reveals a new band of the size of recombination product 1 in the sample treated with Cre recombinase compared to the control. Recombination product 2 is less evident due to its smaller size and mass. According to the instructions from the manufacturer (NEB), the efficiency of in vitro Cre recombination is low for which this assay is a qualitative approximation. Nevertheless, a new band of the expected size of the bigger recombination product was observed validating the designed lox2272-66/71 pair.
    Figure Legend Snippet: Validation of the double mutant site lox2272-66/71 . A. Experimental set up. Fragments were amplified by PCR containing the site lox2272-66 or lox2272-72 . After in vitro recombination mediated by Cre, two recombination products are formed of different length compared to the substrates of recombination. B. Gel electrophoresis run after in vitro recombination reveals a new band of the size of recombination product 1 in the sample treated with Cre recombinase compared to the control. Recombination product 2 is less evident due to its smaller size and mass. According to the instructions from the manufacturer (NEB), the efficiency of in vitro Cre recombination is low for which this assay is a qualitative approximation. Nevertheless, a new band of the expected size of the bigger recombination product was observed validating the designed lox2272-66/71 pair.

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, In Vitro, Nucleic Acid Electrophoresis

    27) Product Images from "In vitro DNA SCRaMbLE"

    Article Title: In vitro DNA SCRaMbLE

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03743-6

    Bottom-up in vitro SCRaMbLE. a Schematic of two independent bottom-up in vitro SCRaMbLE strategies. Left panel—donor fragments each carry a TU and a URA3 gene. The acceptor vector encodes two loxPsym sites (green diamond). Right panel—a loxPsym site is inserted in frame with the URA3 ). Donor fragments are flanked by loxPsym sites. In both cases, the acceptor vector and the pool of donor TUs constructs are mixed with Cre recombinase in vitro. The donor TUs will be randomly inserted into loxPsym sites of the acceptor vector. Note that in bottom-up SCRaMbLE the core β-carotene pathway itself is not in a SCRaMbLE format (i.e., unlike “top down”, there are no lox sites flanking core pathway genes). (yellow box “NonSCRaMbLEable pathway” integrated into genome). Transcription unit (TU), HIS3 auxotroph marker (H), URA3 auxotroph marker (U). b Overview of the carotenoid biosynthetic pathway in S. cerevisiae . Genes shown in black are endogenous to S. cerevisiae . Genes shown in red are non-native, and derive from X. dendrorhous ( crtE , crtYB , crtI ), and one from S. cerevisiae (truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene [ tHMG1 ]). c Yeast colonies transformed with bottom-up in vitro SCRaMbLEd candidate carotenogenic TU pools. The non-SCRaMbLE sample was transformed with the acceptor vector as a control. Three other in vitro SCRaMbLEd pools consisted of endogenous TUs, exogenous TUs, and endogenous + exogenous TUs as indicated. d The efficiency of bottom-up in vitro SCRaMbLE (strategy 1). Different mole ratios of acceptor vector with donor TUs (1:5, 1:10, 1:20, 1:100) were used to test SCRaMbLE efficiency. pYW0113 was used as the acceptor vector; crtI TU and tHMG1 TU were used as donor fragments. A total of 100 yeast colonies for each group were tested using long fragment PCR and restriction enzyme digestion of recovered plasmids
    Figure Legend Snippet: Bottom-up in vitro SCRaMbLE. a Schematic of two independent bottom-up in vitro SCRaMbLE strategies. Left panel—donor fragments each carry a TU and a URA3 gene. The acceptor vector encodes two loxPsym sites (green diamond). Right panel—a loxPsym site is inserted in frame with the URA3 ). Donor fragments are flanked by loxPsym sites. In both cases, the acceptor vector and the pool of donor TUs constructs are mixed with Cre recombinase in vitro. The donor TUs will be randomly inserted into loxPsym sites of the acceptor vector. Note that in bottom-up SCRaMbLE the core β-carotene pathway itself is not in a SCRaMbLE format (i.e., unlike “top down”, there are no lox sites flanking core pathway genes). (yellow box “NonSCRaMbLEable pathway” integrated into genome). Transcription unit (TU), HIS3 auxotroph marker (H), URA3 auxotroph marker (U). b Overview of the carotenoid biosynthetic pathway in S. cerevisiae . Genes shown in black are endogenous to S. cerevisiae . Genes shown in red are non-native, and derive from X. dendrorhous ( crtE , crtYB , crtI ), and one from S. cerevisiae (truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene [ tHMG1 ]). c Yeast colonies transformed with bottom-up in vitro SCRaMbLEd candidate carotenogenic TU pools. The non-SCRaMbLE sample was transformed with the acceptor vector as a control. Three other in vitro SCRaMbLEd pools consisted of endogenous TUs, exogenous TUs, and endogenous + exogenous TUs as indicated. d The efficiency of bottom-up in vitro SCRaMbLE (strategy 1). Different mole ratios of acceptor vector with donor TUs (1:5, 1:10, 1:20, 1:100) were used to test SCRaMbLE efficiency. pYW0113 was used as the acceptor vector; crtI TU and tHMG1 TU were used as donor fragments. A total of 100 yeast colonies for each group were tested using long fragment PCR and restriction enzyme digestion of recovered plasmids

    Techniques Used: In Vitro, Plasmid Preparation, Construct, Marker, Transformation Assay, Polymerase Chain Reaction

    28) Product Images from "Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP"

    Article Title: Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP

    Journal: Biological Procedures Online

    doi: 10.1186/s12575-016-0046-x

    a Schematic of HuEV-A expression. ColE1 ori = bacterial origin of replication; Amp. = Ampicillin resistance cassette; EBNA-1 = Epstein-Barr nuclear antigen 1; OriP = origin of plasmid replication; Gateway = Gateway cloning cassette; Tet-CMV prom. = Doxycycline inducible promoter; FRT = flippase recognition target; FLP = flippase recombinase; LoxP = Lox sequence; Cre = Cre recombinase; TEV = TEV protease cleavage site. Note that treatment with Cre recombinase or FLP recombinase can produce a “short tag” or untagged derivative of the originally cloned ORF, respectively. b Immunoblot of cell lysates from HeLa cells expressing and empty HuEV-A vector or HES1, URI, or Art-27 proteins expressed from the HuEV-A expression vector. A version of the HuEV-A expression vectors not containing YFP in the tag (after Cre treatment of the vector) was also used for each of the proteins and for the empty vector. The proteins were detected with a FLAG-M2 antibody shown in green. Tubulin (shown in red) was used as loading control
    Figure Legend Snippet: a Schematic of HuEV-A expression. ColE1 ori = bacterial origin of replication; Amp. = Ampicillin resistance cassette; EBNA-1 = Epstein-Barr nuclear antigen 1; OriP = origin of plasmid replication; Gateway = Gateway cloning cassette; Tet-CMV prom. = Doxycycline inducible promoter; FRT = flippase recognition target; FLP = flippase recombinase; LoxP = Lox sequence; Cre = Cre recombinase; TEV = TEV protease cleavage site. Note that treatment with Cre recombinase or FLP recombinase can produce a “short tag” or untagged derivative of the originally cloned ORF, respectively. b Immunoblot of cell lysates from HeLa cells expressing and empty HuEV-A vector or HES1, URI, or Art-27 proteins expressed from the HuEV-A expression vector. A version of the HuEV-A expression vectors not containing YFP in the tag (after Cre treatment of the vector) was also used for each of the proteins and for the empty vector. The proteins were detected with a FLAG-M2 antibody shown in green. Tubulin (shown in red) was used as loading control

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Sequencing

    29) Product Images from "An essential cell-autonomous role for hepcidin in cardiac iron homeostasis"

    Article Title: An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

    Journal: eLife

    doi: 10.7554/eLife.19804

    Strategy for generation of Hamp fl/fl ; Myh6.Cre+ mice. A targeting vector was designed to introduce a floxed Hamp allele into mouse ES cells, with exons 2 and 3, which encode the majority of the peptide, flanked by LoxP sites. Further breeding with a C57BL/6 Flp recombinase deleter mouse allowed removal of the Neo cassette. Cardiac Hamp knockouts were then generated by crossing homozygous Hamp fl/fl animals with mice transgenic for Myh6-Cre recombinase, which is under the control of cardiomyocyte-specific Myosin Alpha Heavy chain six promoter. DOI: http://dx.doi.org/10.7554/eLife.19804.026
    Figure Legend Snippet: Strategy for generation of Hamp fl/fl ; Myh6.Cre+ mice. A targeting vector was designed to introduce a floxed Hamp allele into mouse ES cells, with exons 2 and 3, which encode the majority of the peptide, flanked by LoxP sites. Further breeding with a C57BL/6 Flp recombinase deleter mouse allowed removal of the Neo cassette. Cardiac Hamp knockouts were then generated by crossing homozygous Hamp fl/fl animals with mice transgenic for Myh6-Cre recombinase, which is under the control of cardiomyocyte-specific Myosin Alpha Heavy chain six promoter. DOI: http://dx.doi.org/10.7554/eLife.19804.026

    Techniques Used: Mouse Assay, Plasmid Preparation, Introduce, Generated, Transgenic Assay

    Strategy for generation of Slc40a1 C326Y fl/fl ; Myh6.Cre+ mice. A targeting vector was designed to introduce a floxed Slc40a1 C326Y allele into mouse ES cells, containing mutant exon seven and wild type and to delete simultaneously endogenous exons 7 and 8. Further breeding with a C57BL/6 Flp recombinase deleter mouse allowed removal of the Neo cassette. Cardiac Slc40a1 C326Y knock-ins were then generated by crossing homozygous Slc40a1 C326Y fl/fl animals with mice transgenic for Myh6-Cre recombinase, which is under the control of cardiomyocyte-specific Myosin Alpha Heavy chain six promoter. DOI: http://dx.doi.org/10.7554/eLife.19804.027
    Figure Legend Snippet: Strategy for generation of Slc40a1 C326Y fl/fl ; Myh6.Cre+ mice. A targeting vector was designed to introduce a floxed Slc40a1 C326Y allele into mouse ES cells, containing mutant exon seven and wild type and to delete simultaneously endogenous exons 7 and 8. Further breeding with a C57BL/6 Flp recombinase deleter mouse allowed removal of the Neo cassette. Cardiac Slc40a1 C326Y knock-ins were then generated by crossing homozygous Slc40a1 C326Y fl/fl animals with mice transgenic for Myh6-Cre recombinase, which is under the control of cardiomyocyte-specific Myosin Alpha Heavy chain six promoter. DOI: http://dx.doi.org/10.7554/eLife.19804.027

    Techniques Used: Mouse Assay, Plasmid Preparation, Introduce, Mutagenesis, Generated, Transgenic Assay

    30) Product Images from "Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum"

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00273

    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
    Figure Legend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Techniques Used: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

    31) Product Images from "Taok2 Controls Behavioral Response to Ethanol in Mice"

    Article Title: Taok2 Controls Behavioral Response to Ethanol in Mice

    Journal: Genes, brain, and behavior

    doi: 10.1111/j.1601-183X.2012.00834.x

    Targeted disruption of the Taok2 gene in mice (a) Schematic of the conditionally targeted Taok2 allele. The Taok2tm1 allele was generated by introduction of lox P sites flanking exons 2 though 7 of the Taok2 gene. Within intron 7 is a neomycin resistance gene flanked by lox P and frt sites. Upon crossing this line to mice expressing Flpe recombinase, the neomycin selectable marker is removed along with one of the lox P sites to generate the Taok2tm1fl allele. In the presence of Cre recombinase, the remaining lox P sites are recombined into a single lox P site, removing exons 2 though 7 and the translational start site to generate the Taok2tm1Δ allele. (b) Brain sagittal section of a control mouse shows strong expression of TAOK2 by immunohistological analysis in all areas, especially in cortex, hippocampus and striatum. (c) Immunohistological analysis shows reduced TAOK2 expression in Taok2tm1fl/fl;Nes-cre brain. (d) Quantitative PCR analysis of total mouse brain extracts shows near absence of Taok2 transcript in Taok2tm1fl/fl;Nes-cre brain ( n = 4) compared to control mice ( n = 2). (e) Western blot showing comparative loss of TAOK2 protein in total brain lysates of Taok2tm1fl/fl;Nes-cre mice relative to controls. (f) Quantification of anti-TAOK2 signal from (e) comparing Taok2tm1fl/fl;Nes-cre mice ( n = 2) and controls ( n = 2). Lox P sites are denoted by grey chevrons, frt sites by black chevrons. Neo = neomycin resistance gene. The positions of restriction sites BamH1 and BsrB1 used in the generation of the targeting construct are indicated. CTX = cortex; HC = hippocampus; STR = striatum. Error bars are mean ± SEM. Asterisks indicate level of significance (* P
    Figure Legend Snippet: Targeted disruption of the Taok2 gene in mice (a) Schematic of the conditionally targeted Taok2 allele. The Taok2tm1 allele was generated by introduction of lox P sites flanking exons 2 though 7 of the Taok2 gene. Within intron 7 is a neomycin resistance gene flanked by lox P and frt sites. Upon crossing this line to mice expressing Flpe recombinase, the neomycin selectable marker is removed along with one of the lox P sites to generate the Taok2tm1fl allele. In the presence of Cre recombinase, the remaining lox P sites are recombined into a single lox P site, removing exons 2 though 7 and the translational start site to generate the Taok2tm1Δ allele. (b) Brain sagittal section of a control mouse shows strong expression of TAOK2 by immunohistological analysis in all areas, especially in cortex, hippocampus and striatum. (c) Immunohistological analysis shows reduced TAOK2 expression in Taok2tm1fl/fl;Nes-cre brain. (d) Quantitative PCR analysis of total mouse brain extracts shows near absence of Taok2 transcript in Taok2tm1fl/fl;Nes-cre brain ( n = 4) compared to control mice ( n = 2). (e) Western blot showing comparative loss of TAOK2 protein in total brain lysates of Taok2tm1fl/fl;Nes-cre mice relative to controls. (f) Quantification of anti-TAOK2 signal from (e) comparing Taok2tm1fl/fl;Nes-cre mice ( n = 2) and controls ( n = 2). Lox P sites are denoted by grey chevrons, frt sites by black chevrons. Neo = neomycin resistance gene. The positions of restriction sites BamH1 and BsrB1 used in the generation of the targeting construct are indicated. CTX = cortex; HC = hippocampus; STR = striatum. Error bars are mean ± SEM. Asterisks indicate level of significance (* P

    Techniques Used: Mouse Assay, Generated, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Construct

    32) Product Images from "Modular assembly of transposon integratable multigene vectors using RecWay assembly"

    Article Title: Modular assembly of transposon integratable multigene vectors using RecWay assembly

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt115

    Assembly of up to 6 gene vectors using Cre Recombinase. ( a ) Diagram of the two dual DEST Shuttle vectors used to subsequently generate four or six gene vectors after dual LR Clonase reaction. The Cre recombinase LoxP and Lox2722 mutant are represented as black and white triangles, respectively. ( b ) Diagram of vector layout of the insulated versions of the shuttle vectors and final PB dual DEST vectors are also shown. ( c ) Schematic of steps to generate bacterial marker-free six gene transposon vectors using RecWay assembly. Three dual clonase reactions are performed using two shuttle vectors and a single transposon dual DEST vector. I-SceI digestion and self-ligation of shuttle vectors containing the cDNAs/shRNAs of interest is performed followed by retrofitting via Cre recombination of all three dual vectors. Once assembled, vectors can be transformed into FLP recombinase-expressing bacteria to remove the Kan R (kanamycin), Cm R (chloramphenicol) and Spec R (spectinomycin) bacterial markers flanked by unique FRT sites (FRT1, 3 and 14, respectively). The FRT sites and insulators are not shown for simplicity; see Supplementary Figure S1a and b for a more highly detailed schematic representation. ( d ) Seven-day timeline for the assembly of bacterial marker-free six gene transposon vectors using RecWay assembly.
    Figure Legend Snippet: Assembly of up to 6 gene vectors using Cre Recombinase. ( a ) Diagram of the two dual DEST Shuttle vectors used to subsequently generate four or six gene vectors after dual LR Clonase reaction. The Cre recombinase LoxP and Lox2722 mutant are represented as black and white triangles, respectively. ( b ) Diagram of vector layout of the insulated versions of the shuttle vectors and final PB dual DEST vectors are also shown. ( c ) Schematic of steps to generate bacterial marker-free six gene transposon vectors using RecWay assembly. Three dual clonase reactions are performed using two shuttle vectors and a single transposon dual DEST vector. I-SceI digestion and self-ligation of shuttle vectors containing the cDNAs/shRNAs of interest is performed followed by retrofitting via Cre recombination of all three dual vectors. Once assembled, vectors can be transformed into FLP recombinase-expressing bacteria to remove the Kan R (kanamycin), Cm R (chloramphenicol) and Spec R (spectinomycin) bacterial markers flanked by unique FRT sites (FRT1, 3 and 14, respectively). The FRT sites and insulators are not shown for simplicity; see Supplementary Figure S1a and b for a more highly detailed schematic representation. ( d ) Seven-day timeline for the assembly of bacterial marker-free six gene transposon vectors using RecWay assembly.

    Techniques Used: Mutagenesis, Plasmid Preparation, Marker, Ligation, Transformation Assay, Expressing

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    New England Biolabs cre recombinase
    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre <t>recombinase</t> in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
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    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    doi: 10.3389/fnmol.2017.00273

    Figure Lengend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Article Snippet: To differentiate between Fyn expressed in D1R vs. D2R MSNs, we developed a method in which short hairpin RNA (shRNA) that targets a specific gene is expressed only in the presence of Cre-recombinase (Cre).

    Techniques: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

    Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Journal: Scientific Reports

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    doi: 10.1038/s41598-018-31585-1

    Figure Lengend Snippet: Effect of Cre recombinase on the polymerization by and the gene expression of phi29 DNA polymerase. ( a ) Effect of Cre recombinase on polymerization by phi29 DNA polymerase. DNA replication of the original circular DNA (0.71 nM) was performed using purified phi29 DNA polymerase (1 U/μl) and the indicated concentration of Cre recombinase in the standard buffer [0.3 mM each dNTP, 50 mM Tris-HCl (pH 7.8), 5 mM magnesium chloride, 7.5 mM potassium chloride, 0.1 mM dithiothreitol] at 30 °C for 12 h. ( b ) Effect of Cre recombinase on the gene expression of GFP. GFP was expressed using a DNA fragment encoding GFP under the T7 promoter (10 nM) and the indicated concentration of Cre recombinase in the TTcDR reaction mixture. Fluorescence intensity of GFP was measured after incubation at 37 °C for 2 h. The error bars indicate standard deviation (n = 3).

    Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

    Techniques: Expressing, Purification, Concentration Assay, Fluorescence, Incubation, Standard Deviation

    DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Journal: Scientific Reports

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    doi: 10.1038/s41598-018-31585-1

    Figure Lengend Snippet: DNA recombination by Cre recombinase. Two DNA fragments (2.3 kb and 2.8 kb), each of which contains a loxP site, were incubated with the indicated concentrations of Cre recombinase, then subjected to 1% agarose gel electrophoresis and DNA staining with SYBR Green I. The black arrowhead indicates the recombination product. The bands around 1.8 kb (indicated with the arrow) represent a byproduct formed during DNA fragment preparation. The far left lane shows a size maker (λ-HindIII).

    Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

    Techniques: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Journal: Scientific Reports

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    doi: 10.1038/s41598-018-31585-1

    Figure Lengend Snippet: Replication of the reproduced circular DNA. ( a ) Experimental scheme. First, a circular DNA (0.40 nM) was replicated with or without 30 mU/μl Cre recombinase in the transcription and translation-coupled DNA replication (TTcDR) mixture at 30 °C for 16 h. Then, the linear DNA was digested with exonuclease V, and the residual circular DNA was used for the second round of the TTcDR reaction. qPCR was used to measure the DNA concentration after the TTcDR reaction before exonuclease treatment. ( b ) The DNA concentrations of the original DNA. The error bars represent standard deviation (n = 3). ( c ) The DNA concentrations of clone 6-wt-loxP. The error bars represent standard deviation (n = 3).

    Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

    Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Journal: Scientific Reports

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    doi: 10.1038/s41598-018-31585-1

    Figure Lengend Snippet: Effect of Cre recombinase on the transcription and translation-coupled DNA replication (TTcDR) reaction. The TTcDR reaction was performed with each circular DNA (0.40 nM) and the indicated concentration of Cre recombinase at 30 °C for 16 h. The amount of product DNA was measured by qPCR and the fold replication was calculated. The error bars indicate standard deviation (n = 3). The original DNA (Original) or the evolved DNAs (clone 6 and clone 6-wt-loxP) were used.

    Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

    Techniques: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Journal: Scientific Reports

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    doi: 10.1038/s41598-018-31585-1

    Figure Lengend Snippet: In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. ( a ) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. ( b ) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). ( c ) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).

    Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

    Techniques: In Vitro, Synthesized, Polymerase Chain Reaction, Amplification, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Journal: Scientific Reports

    Article Title: Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

    doi: 10.1038/s41598-018-31585-1

    Figure Lengend Snippet: Scheme of the transcription and translation-coupled DNA replication (TTcDR)-recombination system to recursively replicate circular DNA. This system consists of a circular DNA containing the phi29 DNA polymerase gene and a loxP site and a reconstituted Escherichia coli translation system containing T7 RNA polymerase and Cre recombinase. ( i ) The DNA is transcribed to synthesize mRNA, which is then used to translate phi29 DNA polymerase. ( ii ) The translated polymerase initiates rolling-circle replication on the circular DNA to synthesize a long linear single-stranded DNA with tandem repeats of the original circular DNA sequence. The phi29 DNA polymerase also synthesizes the complementary strand of the single-stranded DNA to produce a linear double-stranded DNA. ( iii ) Cre recombinase recombines two loxP sites on the long DNA to produce a circular DNA product.

    Article Snippet: The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C.

    Techniques: Sequencing

    Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation

    Journal: BMC Biotechnology

    Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

    doi: 10.1186/s12896-018-0462-x

    Figure Lengend Snippet: Protocol overview. The input plasmid is first mixed with Cre enzyme in 1× reaction buffer. After 20–30 min of incubation at 37 ° C, the reaction is brought to 70 ° C for 10 min to heat inactivate Cre recombinase. The whole reaction can then be directly used for transformation

    Article Snippet: Next, we tested whether we can further boost cloning efficiency by increasing the concentrations of cre recombinase.

    Techniques: Plasmid Preparation, Incubation, Transformation Assay

    Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion

    Journal: BMC Biotechnology

    Article Title: A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

    doi: 10.1186/s12896-018-0462-x

    Figure Lengend Snippet: Cre recombinase effectively inverted ORF in FLEX plasmid in vitro. a Inversion of ORF in plasmid #1. Top, plasmid diagram. (A1) PstI screening. A 926 bp band by pst1 digestion of R1 can be seen form 5 plasmids (# 2,3,7,8,12). There were no R2 and R3 plasmids (should yield a 758 bp band). Colony #1 had low yield. (A2) Plasmid Map. Shown are five PstI sites and two NcoI sites. (A3) Prediction of PstI digestion. b Inversion of ORF in plasmid #2. Top, plasmid diagram. (B1) NcoI digestion. An 813 bp band by NcoI digestion of R1 can be seen from 4 plasmids (# 5,6,7,8). #11 could be either R2 or R3 plasmids for the presence of 645 bp band. (B2) Plasmid Map. PstI and NcoI sites are shown. (B3) Prediction of NcoI digestion

    Article Snippet: Next, we tested whether we can further boost cloning efficiency by increasing the concentrations of cre recombinase.

    Techniques: Plasmid Preparation, In Vitro