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    Structured Review

    New England Biolabs i ceui
    Method to generate a random deletion library in HIV-1. (A) Overview schematic of method to create a barcoded random deletion library. (1) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. (2) <t>Transposons</t> are excised to linearize the insertion library with a meganuclease. (3) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. (4) The chewed termini are end repaired, dA tailed, and then joined by ligation to a T-tailed 60-bp unique barcode cassette. (B) Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction. TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I ( npt ), flanked by meganuclease restriction sites for I-SceI and <t>I-CeuI</t> and Tn 5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. (C) The HIV-1 molecular clone pNL4-3 is a 14,825-bp plasmid harboring the 9,709-bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and lymphadenopathy associated virus [LAV]). (D) Library insertion, excision, and barcoding details. (1) Circular DNA is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. (2) This creates linear DNA with 4-base 3′ overhangs. Deletions are created by bidirectional chewback. (3) Treatment with two exonucleases (T4 and RecJ f ) creates a population of truncated deletion mutants with ragged ends. (4) Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5′ dephosphorylation and addition of a single 3′ dA. (5) Deletion mutants are religated in the presence of a barcode cassette with single 3′ dT overhangs and 5′ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp. Barcodes are constructed with two primer-binding sites (PBS) on either side of a unique 20-bp sequence (barcode N 20 ). (E) Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL4-3 insertion library shows excisions of the TN5MK transposon (1.4 kb) and upward shift of the supercoiled library versus the undigested library. Lane M, 2-log DNA ladder; 1, undigested insertion library; 2, I-SceI digested insertion library; 3, I-CeuI digested insertion library. (F) Location of TN5MK insertions for a subset of 7,559 transposon integrations (3,844 were unique). (G) Determination of enzymatic chewback rate for deletion size. The chewback rate was determined by treating a 4-kb fragment of linear dsDNA with RecJ f and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time and then halting enzymatic activity. Reactions were performed in triplicates. DNA concentrations were established by quantifying the fluorescence of PicoGreen in a plate reader in comparison to that of a dsDNA standard of known concentration. (H) Validation of deletion library. The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (∅) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1 to 4 are the pNL4-3 insertion library, and lanes 5 to 8 are the pNL4-3 deletion library. (I) pNL4-3 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e., right-tailed) histogram of deletion sizes in pNL4-3, with bins of 100 bp (shown in blue), is well-fit by a gamma distribution (green dashed line). (Inset) Number of deletions detected within each region of the HIV genome. (J) Deletion depth profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-3 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication, and bla, β-lactamase, the resistance marker.
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    1) Product Images from "RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements"

    Article Title: RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements

    Journal: mBio

    doi: 10.1128/mBio.01724-20

    Method to generate a random deletion library in HIV-1. (A) Overview schematic of method to create a barcoded random deletion library. (1) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. (2) Transposons are excised to linearize the insertion library with a meganuclease. (3) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. (4) The chewed termini are end repaired, dA tailed, and then joined by ligation to a T-tailed 60-bp unique barcode cassette. (B) Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction. TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I ( npt ), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn 5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. (C) The HIV-1 molecular clone pNL4-3 is a 14,825-bp plasmid harboring the 9,709-bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and lymphadenopathy associated virus [LAV]). (D) Library insertion, excision, and barcoding details. (1) Circular DNA is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. (2) This creates linear DNA with 4-base 3′ overhangs. Deletions are created by bidirectional chewback. (3) Treatment with two exonucleases (T4 and RecJ f ) creates a population of truncated deletion mutants with ragged ends. (4) Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5′ dephosphorylation and addition of a single 3′ dA. (5) Deletion mutants are religated in the presence of a barcode cassette with single 3′ dT overhangs and 5′ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp. Barcodes are constructed with two primer-binding sites (PBS) on either side of a unique 20-bp sequence (barcode N 20 ). (E) Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL4-3 insertion library shows excisions of the TN5MK transposon (1.4 kb) and upward shift of the supercoiled library versus the undigested library. Lane M, 2-log DNA ladder; 1, undigested insertion library; 2, I-SceI digested insertion library; 3, I-CeuI digested insertion library. (F) Location of TN5MK insertions for a subset of 7,559 transposon integrations (3,844 were unique). (G) Determination of enzymatic chewback rate for deletion size. The chewback rate was determined by treating a 4-kb fragment of linear dsDNA with RecJ f and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time and then halting enzymatic activity. Reactions were performed in triplicates. DNA concentrations were established by quantifying the fluorescence of PicoGreen in a plate reader in comparison to that of a dsDNA standard of known concentration. (H) Validation of deletion library. The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (∅) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1 to 4 are the pNL4-3 insertion library, and lanes 5 to 8 are the pNL4-3 deletion library. (I) pNL4-3 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e., right-tailed) histogram of deletion sizes in pNL4-3, with bins of 100 bp (shown in blue), is well-fit by a gamma distribution (green dashed line). (Inset) Number of deletions detected within each region of the HIV genome. (J) Deletion depth profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-3 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication, and bla, β-lactamase, the resistance marker.
    Figure Legend Snippet: Method to generate a random deletion library in HIV-1. (A) Overview schematic of method to create a barcoded random deletion library. (1) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. (2) Transposons are excised to linearize the insertion library with a meganuclease. (3) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. (4) The chewed termini are end repaired, dA tailed, and then joined by ligation to a T-tailed 60-bp unique barcode cassette. (B) Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction. TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I ( npt ), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn 5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. (C) The HIV-1 molecular clone pNL4-3 is a 14,825-bp plasmid harboring the 9,709-bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and lymphadenopathy associated virus [LAV]). (D) Library insertion, excision, and barcoding details. (1) Circular DNA is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. (2) This creates linear DNA with 4-base 3′ overhangs. Deletions are created by bidirectional chewback. (3) Treatment with two exonucleases (T4 and RecJ f ) creates a population of truncated deletion mutants with ragged ends. (4) Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5′ dephosphorylation and addition of a single 3′ dA. (5) Deletion mutants are religated in the presence of a barcode cassette with single 3′ dT overhangs and 5′ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp. Barcodes are constructed with two primer-binding sites (PBS) on either side of a unique 20-bp sequence (barcode N 20 ). (E) Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL4-3 insertion library shows excisions of the TN5MK transposon (1.4 kb) and upward shift of the supercoiled library versus the undigested library. Lane M, 2-log DNA ladder; 1, undigested insertion library; 2, I-SceI digested insertion library; 3, I-CeuI digested insertion library. (F) Location of TN5MK insertions for a subset of 7,559 transposon integrations (3,844 were unique). (G) Determination of enzymatic chewback rate for deletion size. The chewback rate was determined by treating a 4-kb fragment of linear dsDNA with RecJ f and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time and then halting enzymatic activity. Reactions were performed in triplicates. DNA concentrations were established by quantifying the fluorescence of PicoGreen in a plate reader in comparison to that of a dsDNA standard of known concentration. (H) Validation of deletion library. The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (∅) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1 to 4 are the pNL4-3 insertion library, and lanes 5 to 8 are the pNL4-3 deletion library. (I) pNL4-3 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e., right-tailed) histogram of deletion sizes in pNL4-3, with bins of 100 bp (shown in blue), is well-fit by a gamma distribution (green dashed line). (Inset) Number of deletions detected within each region of the HIV genome. (J) Deletion depth profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-3 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication, and bla, β-lactamase, the resistance marker.

    Techniques Used: In Vitro, Ligation, Plasmid Preparation, De-Phosphorylation Assay, Construct, Binding Assay, Sequencing, Activity Assay, Fluorescence, Concentration Assay, Marker

    Application of RanDeL-seq to map Zika virus (ZIKV) cis elements. (A) pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3′ end of the genome, a self-cleaving hepatitis delta virus ribozyme allows for creation of an authentic 3′ end posttranscription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. (B) Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“Ins.”). (+) and (−) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested completed deletion libraries (lanes 1 to 4) were run next to undigested insertion libraries (lanes 5 and 6). Insertion libraries (lanes 7 to 10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (lanes 11 to 14). (C) Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. (D) Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentages of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high-MOI screen and a wild-type ZIKV control (WT). RT-qPCR data were normalized to an MS2 RNA spike-in. (E) Deletion depth profile of intracellular RNA of 293T cotransfected with the wild-type ZIKV plasmid and the pooled deletion libraries. (F) Deletion depth profile of pMR766(+)L after passage 1. Only deletions in Pr to NS1 can be trans -complemented by wild-type ZIKV. (G) Final map of ZIKV cis - and trans -acting elements after passage 2. The two cis -acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans -acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).
    Figure Legend Snippet: Application of RanDeL-seq to map Zika virus (ZIKV) cis elements. (A) pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3′ end of the genome, a self-cleaving hepatitis delta virus ribozyme allows for creation of an authentic 3′ end posttranscription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. (B) Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“Ins.”). (+) and (−) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested completed deletion libraries (lanes 1 to 4) were run next to undigested insertion libraries (lanes 5 and 6). Insertion libraries (lanes 7 to 10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (lanes 11 to 14). (C) Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. (D) Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentages of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high-MOI screen and a wild-type ZIKV control (WT). RT-qPCR data were normalized to an MS2 RNA spike-in. (E) Deletion depth profile of intracellular RNA of 293T cotransfected with the wild-type ZIKV plasmid and the pooled deletion libraries. (F) Deletion depth profile of pMR766(+)L after passage 1. Only deletions in Pr to NS1 can be trans -complemented by wild-type ZIKV. (G) Final map of ZIKV cis - and trans -acting elements after passage 2. The two cis -acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans -acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).

    Techniques Used: Sequencing, Plasmid Preparation, Quantitative RT-PCR, Transmission Assay

    2) Product Images from "Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures"

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evq056

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.
    Figure Legend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Techniques Used:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.
    Figure Legend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Techniques Used: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.
    Figure Legend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Techniques Used:

    3) Product Images from "Molecular Characterization of a Carbapenem-Hydrolyzing Class A ?-Lactamase, SFC-1, from Serratia fonticola UTAD54"

    Article Title: Molecular Characterization of a Carbapenem-Hydrolyzing Class A ?-Lactamase, SFC-1, from Serratia fonticola UTAD54

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.2321-2324.2004

    Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A β-lactamases of S. fonticola ; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.
    Figure Legend Snippet: Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A β-lactamases of S. fonticola ; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.

    Techniques Used: Generated, Pulsed-Field Gel, Electrophoresis, Hybridization, Lambda DNA Preparation

    4) Product Images from "Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿"

    Article Title: Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00111-10

    Localization of bla CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla CMY-2 (C), and int (D).
    Figure Legend Snippet: Localization of bla CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla CMY-2 (C), and int (D).

    Techniques Used: Pulsed-Field Gel, Electrophoresis

    5) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    6) Product Images from "Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii"

    Article Title: Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evu119

    Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).
    Figure Legend Snippet: Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

    Techniques Used: Electrophoresis, Migration, Staining, Molecular Weight

    7) Product Images from "Detection of SGI1/PGI1 Elements and Resistance to Extended-Spectrum Cephalosporins in Proteae of Animal Origin in France"

    Article Title: Detection of SGI1/PGI1 Elements and Resistance to Extended-Spectrum Cephalosporins in Proteae of Animal Origin in France

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00032

    Chromosomal localization of bla CMY -2 in the relevant P. mirabilis isolates. (A) Whole genomic DNAs of isolates 34381 (lane1), 37665 (lane 2), 38327 (lane 3), 38368 (lane 4), 39165 (lane 5), 39175 (lane 6), 39193 (lane 7), and 39214 (lane 8) were digested with I-Ceu I, and the restricted fragments subjected to PFGE. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to bla CMY -2 (B) , and the 23S rRNA gene (C) . The arrows indicate the bands of interest.
    Figure Legend Snippet: Chromosomal localization of bla CMY -2 in the relevant P. mirabilis isolates. (A) Whole genomic DNAs of isolates 34381 (lane1), 37665 (lane 2), 38327 (lane 3), 38368 (lane 4), 39165 (lane 5), 39175 (lane 6), 39193 (lane 7), and 39214 (lane 8) were digested with I-Ceu I, and the restricted fragments subjected to PFGE. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to bla CMY -2 (B) , and the 23S rRNA gene (C) . The arrows indicate the bands of interest.

    Techniques Used:

    8) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    9) Product Images from "Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †"

    Article Title: Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00687-06

    Generation of the wt Ad19a genome upon Tn removal from B19aT51. (A) Schematic representation of the precise removal of the Tn. The KnR gene (arrow) of the Tn (open double arrows) was removed in vitro by I-SceI/I-CeuI meganuclease double digestion (meganuclease sites are indicated by gray lines) followed by end filling and ligation, generating B19aT51ΔKn. In parallel, a PCR was performed using primers specific to the Tn ends (open arrows) flanked by 40-bp homologies to the target sites in Ad (black and gray boxes). In the forward primer the entire target repeat (12345) was incorporated, whereas only the last 3 bp of the right target repeat were included into the homology region of the reverse primer. Target repeats are indicated on either side of the Tn by black and gray numbers. This Tn-containing PCR fragment was introduced into the B19aT51ΔKn by ET recombination, whereby the orientation of the Tn in the newly generated BAC B19aT51T becomes reversed. (B) Tn removal from B19aT51T. B19aT51T was treated with TnsABC* transposase, which excises the Tn, leaving compatible 3-base-long 5′ overhangs on the BAC ends. Simple ligation reconstitutes the 5-bp wt Ad target sequence, thereby generating a BAC containing the wt Ad19a genome (B19a). (C) Restriction analysis of BAC clones and their derived Ads. The XhoI patterns of B19aT51, B19aT51ΔKn, and B19aT51T are shown in lanes 1 to 3, respectively, with Tn-containing fragments indicated by asterisks. The HindIII pattern of BACs (lanes 5 to 7) and reconstituted viruses (lanes 8 and 9) is also shown. Fragment B-derived bands are indicated by asterisks. A HindIII-PacI double digest of B19a DNA releases the end fragments (C and one of the DD′ fragments) from the vector backbone (black arrowhead), eliminating fragment a (lane 7). BAC-derived Ad19a, Ad19aB; wt Ad19a, Ad19a. M indicates the lane of DNA markers (NEB) with numbers in kilobases.
    Figure Legend Snippet: Generation of the wt Ad19a genome upon Tn removal from B19aT51. (A) Schematic representation of the precise removal of the Tn. The KnR gene (arrow) of the Tn (open double arrows) was removed in vitro by I-SceI/I-CeuI meganuclease double digestion (meganuclease sites are indicated by gray lines) followed by end filling and ligation, generating B19aT51ΔKn. In parallel, a PCR was performed using primers specific to the Tn ends (open arrows) flanked by 40-bp homologies to the target sites in Ad (black and gray boxes). In the forward primer the entire target repeat (12345) was incorporated, whereas only the last 3 bp of the right target repeat were included into the homology region of the reverse primer. Target repeats are indicated on either side of the Tn by black and gray numbers. This Tn-containing PCR fragment was introduced into the B19aT51ΔKn by ET recombination, whereby the orientation of the Tn in the newly generated BAC B19aT51T becomes reversed. (B) Tn removal from B19aT51T. B19aT51T was treated with TnsABC* transposase, which excises the Tn, leaving compatible 3-base-long 5′ overhangs on the BAC ends. Simple ligation reconstitutes the 5-bp wt Ad target sequence, thereby generating a BAC containing the wt Ad19a genome (B19a). (C) Restriction analysis of BAC clones and their derived Ads. The XhoI patterns of B19aT51, B19aT51ΔKn, and B19aT51T are shown in lanes 1 to 3, respectively, with Tn-containing fragments indicated by asterisks. The HindIII pattern of BACs (lanes 5 to 7) and reconstituted viruses (lanes 8 and 9) is also shown. Fragment B-derived bands are indicated by asterisks. A HindIII-PacI double digest of B19a DNA releases the end fragments (C and one of the DD′ fragments) from the vector backbone (black arrowhead), eliminating fragment a (lane 7). BAC-derived Ad19a, Ad19aB; wt Ad19a, Ad19a. M indicates the lane of DNA markers (NEB) with numbers in kilobases.

    Techniques Used: In Vitro, Ligation, Polymerase Chain Reaction, Generated, BAC Assay, Sequencing, Clone Assay, Derivative Assay, Plasmid Preparation

    10) Product Images from "Genomic Diversity of Erwinia carotovora subsp. carotovora and Its Correlation with Virulence"

    Article Title: Genomic Diversity of Erwinia carotovora subsp. carotovora and Its Correlation with Virulence

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.5.3013-3023.2004

    Genomic fingerprints of E. carotovora subsp. carotovora as determined by PFGE. (A) I-CeuI genomic cleavage patterns of representative E. carotovora subsp. carotovora strains. Strains WPP17 and WPP19 had unique chromosomal structures compared to the chromosomal structures of other E. carotovora subsp. carotovora strains. (B) Detection of large plasmids in E. carotovora subsp. carotovora 71 (serogroup 3) and E. carotovora subsp. carotovora 63 (serogroup 9). Undigested genomic DNA was compared to I-CeuI-digested DNA of the same strains. In the left panel, the arrows indicate the positions of the putative plasmids. Electrophoresis was performed by using pulse times of 25 to 45 s at 200 V for 20 h at 12°C. Lanes M contained markers. Ecc, E. carotovora subsp. carotovora ; SG, serogroup.
    Figure Legend Snippet: Genomic fingerprints of E. carotovora subsp. carotovora as determined by PFGE. (A) I-CeuI genomic cleavage patterns of representative E. carotovora subsp. carotovora strains. Strains WPP17 and WPP19 had unique chromosomal structures compared to the chromosomal structures of other E. carotovora subsp. carotovora strains. (B) Detection of large plasmids in E. carotovora subsp. carotovora 71 (serogroup 3) and E. carotovora subsp. carotovora 63 (serogroup 9). Undigested genomic DNA was compared to I-CeuI-digested DNA of the same strains. In the left panel, the arrows indicate the positions of the putative plasmids. Electrophoresis was performed by using pulse times of 25 to 45 s at 200 V for 20 h at 12°C. Lanes M contained markers. Ecc, E. carotovora subsp. carotovora ; SG, serogroup.

    Techniques Used: Electrophoresis

    Construction of an I-CeuI physical map. (A) I-CeuI cleavage patterns of genomic DNA of WPP14 and I-CeuI-restricted derivatives. (B) Circular genome map based on restriction data in panel A. The mapped I-CeuI sites localized seven rrn operons, which were designated rrnA through rrnG , and the flanking junction I-CeuI fragments were numbered 1 through 7 in ascending order (the smallest fragment was fragment 1).
    Figure Legend Snippet: Construction of an I-CeuI physical map. (A) I-CeuI cleavage patterns of genomic DNA of WPP14 and I-CeuI-restricted derivatives. (B) Circular genome map based on restriction data in panel A. The mapped I-CeuI sites localized seven rrn operons, which were designated rrnA through rrnG , and the flanking junction I-CeuI fragments were numbered 1 through 7 in ascending order (the smallest fragment was fragment 1).

    Techniques Used:

    11) Product Images from "Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures"

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evq056

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.
    Figure Legend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Techniques Used:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.
    Figure Legend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Techniques Used: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.
    Figure Legend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Techniques Used:

    12) Product Images from "Integrated Genomic Map from Uropathogenic Escherichia coli J96"

    Article Title: Integrated Genomic Map from Uropathogenic Escherichia coli J96

    Journal: Infection and Immunity

    doi:

    J96 Not I, Bln I, and I- Ceu I native genomic DNA digestion patterns. (A) PFGE of wild-type K-12 strain MG1655 and of wild-type strain J96 genomic DNAs digested with Not I, Bln I, and I- Ceu I. PFGE pulse times were ramped from 55 to 65 s over 7 h and from 20 to 30 s over 8 h. (B) Schematic representation of the restriction patterns in panel A. Alphabetical labeling of fragments follows the precedents set in K-12 mapping for Not I, Bln I, and I- Ceu I digests. Individual fragment sizes are to the right in kilobases, with the totals beneath in kilobases. The J96 plasmid band, Q N , is denoted with the superscript P and is found only in native genomic Not I digests. Fragments highlighted with the single asterisk, F N and E B , and the double asterisks, A N and B B , are those containing known loci for J96 pathogenicity islands, Pai-4 and Pai-5 (also called Pai-I and Pai-II, respectively) at 64 and 85 min, respectively.
    Figure Legend Snippet: J96 Not I, Bln I, and I- Ceu I native genomic DNA digestion patterns. (A) PFGE of wild-type K-12 strain MG1655 and of wild-type strain J96 genomic DNAs digested with Not I, Bln I, and I- Ceu I. PFGE pulse times were ramped from 55 to 65 s over 7 h and from 20 to 30 s over 8 h. (B) Schematic representation of the restriction patterns in panel A. Alphabetical labeling of fragments follows the precedents set in K-12 mapping for Not I, Bln I, and I- Ceu I digests. Individual fragment sizes are to the right in kilobases, with the totals beneath in kilobases. The J96 plasmid band, Q N , is denoted with the superscript P and is found only in native genomic Not I digests. Fragments highlighted with the single asterisk, F N and E B , and the double asterisks, A N and B B , are those containing known loci for J96 pathogenicity islands, Pai-4 and Pai-5 (also called Pai-I and Pai-II, respectively) at 64 and 85 min, respectively.

    Techniques Used: Labeling, Plasmid Preparation

    13) Product Images from "Physical and Genetic Map of the Pasteurella multocida A:1 Chromosome"

    Article Title: Physical and Genetic Map of the Pasteurella multocida A:1 Chromosome

    Journal: Journal of Bacteriology

    doi:

    (A) PFGE of P. multocida DNA fragments produced after digestion with Ceu I (lane 1), Not I/ Ceu I (lane 2), Not I (lane 3), Not I/ Apa I (lane 4), Apa I (lane 5), and Apa I/ Ceu I (lane 6). The positions of standard DNA size markers (in kilobases) are shown on the
    Figure Legend Snippet: (A) PFGE of P. multocida DNA fragments produced after digestion with Ceu I (lane 1), Not I/ Ceu I (lane 2), Not I (lane 3), Not I/ Apa I (lane 4), Apa I (lane 5), and Apa I/ Ceu I (lane 6). The positions of standard DNA size markers (in kilobases) are shown on the

    Techniques Used: Produced

    (A) Physical and genetic map of the 2.35-Mbp circular chromosome of P. multocida A:1 PBA100. The positions of the Apa I, Ceu I, and Not I restriction sites are shown, and fragment names are indicated. The genes are positioned on the map to the minimum region
    Figure Legend Snippet: (A) Physical and genetic map of the 2.35-Mbp circular chromosome of P. multocida A:1 PBA100. The positions of the Apa I, Ceu I, and Not I restriction sites are shown, and fragment names are indicated. The genes are positioned on the map to the minimum region

    Techniques Used:

    14) Product Images from "Molecular Epidemiology of Vibrio cholerae O139 in China: Polymorphism of Ribotypes and CTX Elements"

    Article Title: Molecular Epidemiology of Vibrio cholerae O139 in China: Polymorphism of Ribotypes and CTX Elements

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.6.2306-2310.2003

    PFGE profiles (left) of the genomic DNAs of V. cholerae strains belonging to different serogroups and biotypes digested with I- Ceu I and schematic diagram (right) of dendrogram analysis of the strains. Lane 1, 94001 (O139; cholera toxin negative; China); lane 2, 1837 (O139; ctxAB + ; Bangladesh); lane 3, MO45 (O139; ctxAB + ; India); lane 4, 22s (O22; cholera toxin negative; China); lane 5, 86015 (O1; El Tor biotype; cholera toxin negative; China); lane 6, Bin-43 (O1; El Tor biotype; ctxAB + ; China); lane 7, Wujiang-2 (O1; El Tor biotype; ctxAB + ; China); lane 8, 1119 (O1; classical biotype; ctxAB + ; India); lane 9, O395 (O1; classical biotype; ctxAB + ; Bangladesh); lane 10, 569B (O1; classical biotype; ctxAB + ; Bangladesh); lane M, λ DNA ladder molecular size standard.
    Figure Legend Snippet: PFGE profiles (left) of the genomic DNAs of V. cholerae strains belonging to different serogroups and biotypes digested with I- Ceu I and schematic diagram (right) of dendrogram analysis of the strains. Lane 1, 94001 (O139; cholera toxin negative; China); lane 2, 1837 (O139; ctxAB + ; Bangladesh); lane 3, MO45 (O139; ctxAB + ; India); lane 4, 22s (O22; cholera toxin negative; China); lane 5, 86015 (O1; El Tor biotype; cholera toxin negative; China); lane 6, Bin-43 (O1; El Tor biotype; ctxAB + ; China); lane 7, Wujiang-2 (O1; El Tor biotype; ctxAB + ; China); lane 8, 1119 (O1; classical biotype; ctxAB + ; India); lane 9, O395 (O1; classical biotype; ctxAB + ; Bangladesh); lane 10, 569B (O1; classical biotype; ctxAB + ; Bangladesh); lane M, λ DNA ladder molecular size standard.

    Techniques Used:

    15) Product Images from "Chromosome-Encoded Class D ?-Lactamase OXA-23 in Proteus mirabilis"

    Article Title: Chromosome-Encoded Class D ?-Lactamase OXA-23 in Proteus mirabilis

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.46.6.2004-2006.2002

    Localization of bla OXA-23 in I- Ceu I-generated chromosome fragments of P. mirabilis CFO239 separated by PFGE. (A) Chromosome restriction patterns. (B) Hybridization of restricted patterns with a probe specific to 23S rRNA genes. (C) Hybridization of restricted patterns with a probe specific to the bla OXA-23 gene. Lanes: 1, clinical P. mirabilis without the bla OXA-23 gene; 2, OXA-23-producing P. mirabilis CFO239.
    Figure Legend Snippet: Localization of bla OXA-23 in I- Ceu I-generated chromosome fragments of P. mirabilis CFO239 separated by PFGE. (A) Chromosome restriction patterns. (B) Hybridization of restricted patterns with a probe specific to 23S rRNA genes. (C) Hybridization of restricted patterns with a probe specific to the bla OXA-23 gene. Lanes: 1, clinical P. mirabilis without the bla OXA-23 gene; 2, OXA-23-producing P. mirabilis CFO239.

    Techniques Used: Generated, Hybridization

    16) Product Images from "Resurgent Vibrio cholerae O139: Rearrangement of Cholera Toxin Genetic Elements and Amplification of rrn Operon †"

    Article Title: Resurgent Vibrio cholerae O139: Rearrangement of Cholera Toxin Genetic Elements and Amplification of rrn Operon †

    Journal: Infection and Immunity

    doi:

    PFGE of I- Ceu I-digested genomes of V. cholerae O139R and O139B strains. (a) The genomes of representative O139R and O139B strains were digested with I- Ceu I and electrophoresed with a pulse time interpolated between 5 and 100 s for 24 h at 10 V/cm at 3°C, and the gels were stained with ethidium bromide. (b) Autoradiogram of I- Ceu I-digested, end-labeled, PFGE-separated genomic DNA of V. cholerae O139R and O139B. The pulse time described in panel a was followed by ramping between 10 and 150 s for another 12 h at 5 V/cm. Two more I- Ceu I fragments in the O139R genome and one more in the O139B genome could be resolved under these pulse conditions by autoradiography. The numbers represent the I- Ceu I fragment sizes in kilobases.
    Figure Legend Snippet: PFGE of I- Ceu I-digested genomes of V. cholerae O139R and O139B strains. (a) The genomes of representative O139R and O139B strains were digested with I- Ceu I and electrophoresed with a pulse time interpolated between 5 and 100 s for 24 h at 10 V/cm at 3°C, and the gels were stained with ethidium bromide. (b) Autoradiogram of I- Ceu I-digested, end-labeled, PFGE-separated genomic DNA of V. cholerae O139R and O139B. The pulse time described in panel a was followed by ramping between 10 and 150 s for another 12 h at 5 V/cm. Two more I- Ceu I fragments in the O139R genome and one more in the O139B genome could be resolved under these pulse conditions by autoradiography. The numbers represent the I- Ceu I fragment sizes in kilobases.

    Techniques Used: Staining, Labeling, Autoradiography

    17) Product Images from "Genetic Characterization of Atypical Citrobacter freundii"

    Article Title: Genetic Characterization of Atypical Citrobacter freundii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074120

    Pulsed-field gel electrophoresis profiles of l- Ceu I restriction fragments from C. freundii isolates. a) Pulsed-field gel shows Ceu I profiles: line 1, C. freundii E9750 NCTC; lane 2, FMU108327/P; lane 3, FMU108327/A 10 and lane 4; S. Typhimurium LT2 ATCC 700720. The results of Southern blotting analysis appear with capital letters. These indicate the Ceús individual fragments of C. freundii corresponding to those of S . Typhimurium LT2; b) chromosome sizes and molecular weights of individual Ceu I fragments. It also shows the location of every marker gene according to its correspondence in each Ceu I fragment. The Ceu I-C and Ceu I-F fragments of C. freundii were determined by probing the isolated DNA from S. Typhimurium LT2 Ceu I-C and Ceu I-F fragments; c) the gel from panel a were blotted to N+nylon membranes and probed as follows: lanes 1–4 met H ( Table S1 ); lanes 5–8 DNA from C. freundii fragment Ceu I-H and lanes 9–12, met A. Ceu I fragments identified by the probes are indicated inside the images with capital letters.
    Figure Legend Snippet: Pulsed-field gel electrophoresis profiles of l- Ceu I restriction fragments from C. freundii isolates. a) Pulsed-field gel shows Ceu I profiles: line 1, C. freundii E9750 NCTC; lane 2, FMU108327/P; lane 3, FMU108327/A 10 and lane 4; S. Typhimurium LT2 ATCC 700720. The results of Southern blotting analysis appear with capital letters. These indicate the Ceús individual fragments of C. freundii corresponding to those of S . Typhimurium LT2; b) chromosome sizes and molecular weights of individual Ceu I fragments. It also shows the location of every marker gene according to its correspondence in each Ceu I fragment. The Ceu I-C and Ceu I-F fragments of C. freundii were determined by probing the isolated DNA from S. Typhimurium LT2 Ceu I-C and Ceu I-F fragments; c) the gel from panel a were blotted to N+nylon membranes and probed as follows: lanes 1–4 met H ( Table S1 ); lanes 5–8 DNA from C. freundii fragment Ceu I-H and lanes 9–12, met A. Ceu I fragments identified by the probes are indicated inside the images with capital letters.

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Southern Blot, Marker, Isolation

    18) Product Images from "Macrolide Resistance Gene mreA of Streptococcus agalactiae Encodes a Flavokinase"

    Article Title: Macrolide Resistance Gene mreA of Streptococcus agalactiae Encodes a Flavokinase

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.45.8.2280-2286.2001

    Analysis of genomic DNA from S. agalactiae COH31 γ/δ (lane 1) and UCN4 (lane 2), digested with I -Ceu I by pulsed-field gel electrophoresis (left) and hybridization (middle and right). The digested fragments were transferred to a nylon sheet and hybridized to an in vitro digoxigenin-labeled 16S probe (middle). After dehybridization, the filter was hybridized to a digoxigenin-labeled mreA probe (right).
    Figure Legend Snippet: Analysis of genomic DNA from S. agalactiae COH31 γ/δ (lane 1) and UCN4 (lane 2), digested with I -Ceu I by pulsed-field gel electrophoresis (left) and hybridization (middle and right). The digested fragments were transferred to a nylon sheet and hybridized to an in vitro digoxigenin-labeled 16S probe (middle). After dehybridization, the filter was hybridized to a digoxigenin-labeled mreA probe (right).

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Hybridization, In Vitro, Labeling

    19) Product Images from "Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens"

    Article Title: Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044449

    PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant C. perfringens strain c1261_A. PFGE analysis of C. perfringens strain c1261_A total DNA (A). Southern blot of C. perfringens isolate c1261_A total DNA probed with rrn (B) and with bcrB (C). Sizes (in kilobases) are indicated on the left.
    Figure Legend Snippet: PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant C. perfringens strain c1261_A. PFGE analysis of C. perfringens strain c1261_A total DNA (A). Southern blot of C. perfringens isolate c1261_A total DNA probed with rrn (B) and with bcrB (C). Sizes (in kilobases) are indicated on the left.

    Techniques Used: Hybridization, Southern Blot

    20) Product Images from "RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements"

    Article Title: RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

    Journal: bioRxiv

    doi: 10.1101/2020.07.01.183574

    Application of RanDeL-seq to map Zika Virus (ZIKV) cis elements. A. pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3’ end of the genome, a self-cleaving Hepatitis Delta ribozyme allows for creation of an authentic 3’ end post-transcription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. B. Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“ins.”). (+) and (-) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested, completed deletion libraries (Lanes 1-4) were run next to undigested insertion libraries (Lanes 5-6). Insertion libraries (Lanes 7-10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (Lanes 11-14). C. Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well-represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. D. Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentage of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high MOI screen, and a wild-type ZIKV control (WT). RT-qPCR data was normalized to a MS2 RNA spike-in. E. Deletion Depth Profile of intracellular RNA of 293T co-transfected with the wild-type ZIKV plasmid and the pooled deletion libraries. F. Deletion Depth Profile of pMR766(+)L after Passage 1. Only deletions in Pr–NS1 can be trans-complemented by wild-type ZIKV. H. Final map of ZIKV cis- and trans-acting elements after Passage 2. The two cis-acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans-acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).
    Figure Legend Snippet: Application of RanDeL-seq to map Zika Virus (ZIKV) cis elements. A. pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3’ end of the genome, a self-cleaving Hepatitis Delta ribozyme allows for creation of an authentic 3’ end post-transcription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. B. Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“ins.”). (+) and (-) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested, completed deletion libraries (Lanes 1-4) were run next to undigested insertion libraries (Lanes 5-6). Insertion libraries (Lanes 7-10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (Lanes 11-14). C. Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well-represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. D. Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentage of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high MOI screen, and a wild-type ZIKV control (WT). RT-qPCR data was normalized to a MS2 RNA spike-in. E. Deletion Depth Profile of intracellular RNA of 293T co-transfected with the wild-type ZIKV plasmid and the pooled deletion libraries. F. Deletion Depth Profile of pMR766(+)L after Passage 1. Only deletions in Pr–NS1 can be trans-complemented by wild-type ZIKV. H. Final map of ZIKV cis- and trans-acting elements after Passage 2. The two cis-acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans-acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).

    Techniques Used: Sequencing, Plasmid Preparation, Quantitative RT-PCR, Transfection, Transmission Assay

    Method to generate a Random Deletion Library in HIV-1 A. Overview schematic of method to create a barcoded random deletion library: ( 1 ) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. ( 2 ) Transposons are excised to linearize the insertion library with a meganuclease. ( 3 ) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. ( 4 ) The chewed termini are end-repaired, dA-tailed, then joined by ligation to a T-tailed 60bp unique barcode cassette. B. Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction: TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I (npt), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. C. The HIV-1 molecular clone pNL4-3, is a 14825 bp plasmid harboring the 9709 bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and LAV). D.Library insertion, excision, barcoding details: Circular DNA ( 1 ) is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. This creates linear DNA with 4 base 3’ overhangs ( 2 ). Deletions are created by bidirectional chewback. Treatment with two exonucleases (T4 and RecJf) creates a population of truncated deletion mutants with ragged ends ( 3 ). Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5’ dephosphorylation and addition of a single 3’-dA ( 4 ). Deletion mutants are re-ligated in presence of a barcode cassette with single 3’-dT overhangs and 5’ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp ( 5 ). E. Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL43 insertion library shows excisions of the TN5MK transposon (1.4kb) and upward shift of the supercoiled library vs. the undigested library. Lanes: (M) 2 log DNA ladder, ( 1 ) undigested insertion library, ( 2 ) I-SceI digested insertion library, ( 3 ) I-CeuI digested insertion library. F. Location of TN5MK insertions for a subset of 7559 transposon integrations (3844 were unique). G.Determination of enzymatic chewback rate for deletion size: The chewback rate was determined by treating a 4 kb fragment of linear dsDNA with RecJf and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time, then halting enzymatic activity. Reactions were performed in triplicate. DNA concentrations were established by quantifying fluorescence of PicoGreen in a plate reader in comparison to a dsDNA standard of known concentration. H.Validation of Deletion Library: The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (Ø) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1-4 are the pNL43 insertion library and Lanes 5-8 are the pNL43 deletion library. I.pNL4-31 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e. right-tailed) histogram of deletion sizes in pNL4-31, with bins of 100 bp (shown in blue) is well-fit by a Gamma distribution (green, broken-line). Inset: Number of deletions detected within each region of the HIV genome. J.Deletion Depth Profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-31 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication and bla, -lactamase, the resistance marker
    Figure Legend Snippet: Method to generate a Random Deletion Library in HIV-1 A. Overview schematic of method to create a barcoded random deletion library: ( 1 ) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. ( 2 ) Transposons are excised to linearize the insertion library with a meganuclease. ( 3 ) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. ( 4 ) The chewed termini are end-repaired, dA-tailed, then joined by ligation to a T-tailed 60bp unique barcode cassette. B. Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction: TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I (npt), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. C. The HIV-1 molecular clone pNL4-3, is a 14825 bp plasmid harboring the 9709 bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and LAV). D.Library insertion, excision, barcoding details: Circular DNA ( 1 ) is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. This creates linear DNA with 4 base 3’ overhangs ( 2 ). Deletions are created by bidirectional chewback. Treatment with two exonucleases (T4 and RecJf) creates a population of truncated deletion mutants with ragged ends ( 3 ). Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5’ dephosphorylation and addition of a single 3’-dA ( 4 ). Deletion mutants are re-ligated in presence of a barcode cassette with single 3’-dT overhangs and 5’ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp ( 5 ). E. Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL43 insertion library shows excisions of the TN5MK transposon (1.4kb) and upward shift of the supercoiled library vs. the undigested library. Lanes: (M) 2 log DNA ladder, ( 1 ) undigested insertion library, ( 2 ) I-SceI digested insertion library, ( 3 ) I-CeuI digested insertion library. F. Location of TN5MK insertions for a subset of 7559 transposon integrations (3844 were unique). G.Determination of enzymatic chewback rate for deletion size: The chewback rate was determined by treating a 4 kb fragment of linear dsDNA with RecJf and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time, then halting enzymatic activity. Reactions were performed in triplicate. DNA concentrations were established by quantifying fluorescence of PicoGreen in a plate reader in comparison to a dsDNA standard of known concentration. H.Validation of Deletion Library: The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (Ø) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1-4 are the pNL43 insertion library and Lanes 5-8 are the pNL43 deletion library. I.pNL4-31 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e. right-tailed) histogram of deletion sizes in pNL4-31, with bins of 100 bp (shown in blue) is well-fit by a Gamma distribution (green, broken-line). Inset: Number of deletions detected within each region of the HIV genome. J.Deletion Depth Profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-31 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication and bla, -lactamase, the resistance marker

    Techniques Used: In Vitro, Ligation, Plasmid Preparation, De-Phosphorylation Assay, Activity Assay, Fluorescence, Concentration Assay, Marker

    21) Product Images from "Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-?-Lactamase Gene, blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia"

    Article Title: Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-?-Lactamase Gene, blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.05654-11

    Evidence of movement of IS CR15 and bla AIM-1 , and relatedness of AIM-1-positive strains. All panels a marker (lane M), WCH2677 (lane 1), WCH2813 (lane 2), and WCH2837 (lane 3). (A) SpeI digestion. (B) I-CeuI digestion. (C) S1 digestion and probing with
    Figure Legend Snippet: Evidence of movement of IS CR15 and bla AIM-1 , and relatedness of AIM-1-positive strains. All panels a marker (lane M), WCH2677 (lane 1), WCH2813 (lane 2), and WCH2837 (lane 3). (A) SpeI digestion. (B) I-CeuI digestion. (C) S1 digestion and probing with

    Techniques Used: Marker

    22) Product Images from "VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants ▿"

    Article Title: VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants ▿

    Journal:

    doi: 10.1128/AEM.02239-06

    PFGE of S1-digested (A) and I-CeuI-digested (C) total DNA and corresponding vanA (B and E) and 16S rRNA gene (D) hybridization. Lane 1, E. faecium HI-MI28; lane 2, E. faecium HI-MI34; lane 3, E. faecium HI-MI60; lane 4, E. faecium HI-MI32; lane 5, E.
    Figure Legend Snippet: PFGE of S1-digested (A) and I-CeuI-digested (C) total DNA and corresponding vanA (B and E) and 16S rRNA gene (D) hybridization. Lane 1, E. faecium HI-MI28; lane 2, E. faecium HI-MI34; lane 3, E. faecium HI-MI60; lane 4, E. faecium HI-MI32; lane 5, E.

    Techniques Used: Hybridization

    23) Product Images from "A recombineering based approach for high-throughput conditional knockout targeting vector construction"

    Article Title: A recombineering based approach for high-throughput conditional knockout targeting vector construction

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm163

    Construction of conditional knockout targeting vectors using the new recombineering reagents. ( A ) The genomic structure of a locus with exons 3–5 to be deleted in the cko allele. The Bsd cassette flanked by two rare cutter sites, I-SceI and I-CeuI, is targeted to the 5′ side of the intended deletion region. Subsequently, the loxP-F3-PGK-EM7-Neo-F3 ( Neo ) cassette is targeted to the 3′ side of the deletion region. The point mutation present in the Neo coding sequence of PL452 and PL451 plasmids ( 10 , 36 ) was corrected in this Neo cassette which resulted in higher resistance to Kanamycin in E. coli and a 2-fold increase in the number of G418-resistant ES colonies. Coloured lines represent the short homology arms in recombineering. ( B ) The genomic DNA fragment is retrieved from the BAC to PL611, which has the Amp R gene. In a typical cko vector, we choose 4–5-kb genomic DNA as the left homology arm (5′), and 2–3 kb as the right homology arm (3′). The genomic DNA region to be deleted is generally between 1 and 7 kb. ( C ) The Bsd cassette can conveniently be replaced by a reporter, i.e. lacZ , in a simple ligation reaction. The final targeting vector has the reporter flanked by two FRT sites followed by a lox P site at the 5′ side of the intended deletion region, and a F3 flanked Neo cassette providing positive selection in ES cells. The negative selection marker TK is added to the vector backbone by recombineering. The vector is linearized with the rare-cutter I-PpoI.
    Figure Legend Snippet: Construction of conditional knockout targeting vectors using the new recombineering reagents. ( A ) The genomic structure of a locus with exons 3–5 to be deleted in the cko allele. The Bsd cassette flanked by two rare cutter sites, I-SceI and I-CeuI, is targeted to the 5′ side of the intended deletion region. Subsequently, the loxP-F3-PGK-EM7-Neo-F3 ( Neo ) cassette is targeted to the 3′ side of the deletion region. The point mutation present in the Neo coding sequence of PL452 and PL451 plasmids ( 10 , 36 ) was corrected in this Neo cassette which resulted in higher resistance to Kanamycin in E. coli and a 2-fold increase in the number of G418-resistant ES colonies. Coloured lines represent the short homology arms in recombineering. ( B ) The genomic DNA fragment is retrieved from the BAC to PL611, which has the Amp R gene. In a typical cko vector, we choose 4–5-kb genomic DNA as the left homology arm (5′), and 2–3 kb as the right homology arm (3′). The genomic DNA region to be deleted is generally between 1 and 7 kb. ( C ) The Bsd cassette can conveniently be replaced by a reporter, i.e. lacZ , in a simple ligation reaction. The final targeting vector has the reporter flanked by two FRT sites followed by a lox P site at the 5′ side of the intended deletion region, and a F3 flanked Neo cassette providing positive selection in ES cells. The negative selection marker TK is added to the vector backbone by recombineering. The vector is linearized with the rare-cutter I-PpoI.

    Techniques Used: Knock-Out, Mutagenesis, Sequencing, BAC Assay, Plasmid Preparation, Ligation, Selection, Marker

    24) Product Images from "Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii"

    Article Title: Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evu119

    Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).
    Figure Legend Snippet: Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

    Techniques Used: Electrophoresis, Migration, Staining, Molecular Weight

    25) Product Images from "Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures"

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evq056

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.
    Figure Legend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Techniques Used:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.
    Figure Legend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Techniques Used: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.
    Figure Legend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Techniques Used:

    26) Product Images from "Salmonella genomic island 3 is an integrative and conjugative element and contributes to copper and arsenic resistance of Salmonella enterica"

    Article Title: Salmonella genomic island 3 is an integrative and conjugative element and contributes to copper and arsenic resistance of Salmonella enterica

    Journal: bioRxiv

    doi: 10.1101/564534

    PCR and PFGE-Southern blot hybridization images demonstrating the chromosomal location and/or generation of the circular form of SGI3 in Salmonella 4,[5],12:i:-strains and Salmonella Typhimurium LT2 transconjugants. Lane 1, L-3838; lane 2, L-3841; lane 3, LT2; lane 4, LT2TC pheV ; lane 5, LT2TC pheR ; lane M1, 100-bp DNA ladder; lane M2, XbaI-digested Salmonella Braenderup. (a) SNP genotyping and PCR to detect junction regions of integrated or circular form of SGI3. (b) PFGE separation of I-CeuI-digested genomic DNA from S. enterica strains followed by Southern blot hybridization with 23S rRNA, integrase, and pcoA gene probes
    Figure Legend Snippet: PCR and PFGE-Southern blot hybridization images demonstrating the chromosomal location and/or generation of the circular form of SGI3 in Salmonella 4,[5],12:i:-strains and Salmonella Typhimurium LT2 transconjugants. Lane 1, L-3838; lane 2, L-3841; lane 3, LT2; lane 4, LT2TC pheV ; lane 5, LT2TC pheR ; lane M1, 100-bp DNA ladder; lane M2, XbaI-digested Salmonella Braenderup. (a) SNP genotyping and PCR to detect junction regions of integrated or circular form of SGI3. (b) PFGE separation of I-CeuI-digested genomic DNA from S. enterica strains followed by Southern blot hybridization with 23S rRNA, integrase, and pcoA gene probes

    Techniques Used: Polymerase Chain Reaction, Southern Blot, Hybridization, Genotyping Assay

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    New England Biolabs i ceui
    Method to generate a random deletion library in HIV-1. (A) Overview schematic of method to create a barcoded random deletion library. (1) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. (2) <t>Transposons</t> are excised to linearize the insertion library with a meganuclease. (3) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. (4) The chewed termini are end repaired, dA tailed, and then joined by ligation to a T-tailed 60-bp unique barcode cassette. (B) Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction. TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I ( npt ), flanked by meganuclease restriction sites for I-SceI and <t>I-CeuI</t> and Tn 5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. (C) The HIV-1 molecular clone pNL4-3 is a 14,825-bp plasmid harboring the 9,709-bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and lymphadenopathy associated virus [LAV]). (D) Library insertion, excision, and barcoding details. (1) Circular DNA is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. (2) This creates linear DNA with 4-base 3′ overhangs. Deletions are created by bidirectional chewback. (3) Treatment with two exonucleases (T4 and RecJ f ) creates a population of truncated deletion mutants with ragged ends. (4) Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5′ dephosphorylation and addition of a single 3′ dA. (5) Deletion mutants are religated in the presence of a barcode cassette with single 3′ dT overhangs and 5′ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp. Barcodes are constructed with two primer-binding sites (PBS) on either side of a unique 20-bp sequence (barcode N 20 ). (E) Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL4-3 insertion library shows excisions of the TN5MK transposon (1.4 kb) and upward shift of the supercoiled library versus the undigested library. Lane M, 2-log DNA ladder; 1, undigested insertion library; 2, I-SceI digested insertion library; 3, I-CeuI digested insertion library. (F) Location of TN5MK insertions for a subset of 7,559 transposon integrations (3,844 were unique). (G) Determination of enzymatic chewback rate for deletion size. The chewback rate was determined by treating a 4-kb fragment of linear dsDNA with RecJ f and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time and then halting enzymatic activity. Reactions were performed in triplicates. DNA concentrations were established by quantifying the fluorescence of PicoGreen in a plate reader in comparison to that of a dsDNA standard of known concentration. (H) Validation of deletion library. The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (∅) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1 to 4 are the pNL4-3 insertion library, and lanes 5 to 8 are the pNL4-3 deletion library. (I) pNL4-3 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e., right-tailed) histogram of deletion sizes in pNL4-3, with bins of 100 bp (shown in blue), is well-fit by a gamma distribution (green dashed line). (Inset) Number of deletions detected within each region of the HIV genome. (J) Deletion depth profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-3 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication, and bla, β-lactamase, the resistance marker.
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    Method to generate a random deletion library in HIV-1. (A) Overview schematic of method to create a barcoded random deletion library. (1) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. (2) Transposons are excised to linearize the insertion library with a meganuclease. (3) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. (4) The chewed termini are end repaired, dA tailed, and then joined by ligation to a T-tailed 60-bp unique barcode cassette. (B) Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction. TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I ( npt ), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn 5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. (C) The HIV-1 molecular clone pNL4-3 is a 14,825-bp plasmid harboring the 9,709-bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and lymphadenopathy associated virus [LAV]). (D) Library insertion, excision, and barcoding details. (1) Circular DNA is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. (2) This creates linear DNA with 4-base 3′ overhangs. Deletions are created by bidirectional chewback. (3) Treatment with two exonucleases (T4 and RecJ f ) creates a population of truncated deletion mutants with ragged ends. (4) Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5′ dephosphorylation and addition of a single 3′ dA. (5) Deletion mutants are religated in the presence of a barcode cassette with single 3′ dT overhangs and 5′ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp. Barcodes are constructed with two primer-binding sites (PBS) on either side of a unique 20-bp sequence (barcode N 20 ). (E) Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL4-3 insertion library shows excisions of the TN5MK transposon (1.4 kb) and upward shift of the supercoiled library versus the undigested library. Lane M, 2-log DNA ladder; 1, undigested insertion library; 2, I-SceI digested insertion library; 3, I-CeuI digested insertion library. (F) Location of TN5MK insertions for a subset of 7,559 transposon integrations (3,844 were unique). (G) Determination of enzymatic chewback rate for deletion size. The chewback rate was determined by treating a 4-kb fragment of linear dsDNA with RecJ f and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time and then halting enzymatic activity. Reactions were performed in triplicates. DNA concentrations were established by quantifying the fluorescence of PicoGreen in a plate reader in comparison to that of a dsDNA standard of known concentration. (H) Validation of deletion library. The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (∅) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1 to 4 are the pNL4-3 insertion library, and lanes 5 to 8 are the pNL4-3 deletion library. (I) pNL4-3 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e., right-tailed) histogram of deletion sizes in pNL4-3, with bins of 100 bp (shown in blue), is well-fit by a gamma distribution (green dashed line). (Inset) Number of deletions detected within each region of the HIV genome. (J) Deletion depth profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-3 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication, and bla, β-lactamase, the resistance marker.

    Journal: mBio

    Article Title: RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements

    doi: 10.1128/mBio.01724-20

    Figure Lengend Snippet: Method to generate a random deletion library in HIV-1. (A) Overview schematic of method to create a barcoded random deletion library. (1) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. (2) Transposons are excised to linearize the insertion library with a meganuclease. (3) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. (4) The chewed termini are end repaired, dA tailed, and then joined by ligation to a T-tailed 60-bp unique barcode cassette. (B) Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction. TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I ( npt ), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn 5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. (C) The HIV-1 molecular clone pNL4-3 is a 14,825-bp plasmid harboring the 9,709-bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and lymphadenopathy associated virus [LAV]). (D) Library insertion, excision, and barcoding details. (1) Circular DNA is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. (2) This creates linear DNA with 4-base 3′ overhangs. Deletions are created by bidirectional chewback. (3) Treatment with two exonucleases (T4 and RecJ f ) creates a population of truncated deletion mutants with ragged ends. (4) Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5′ dephosphorylation and addition of a single 3′ dA. (5) Deletion mutants are religated in the presence of a barcode cassette with single 3′ dT overhangs and 5′ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp. Barcodes are constructed with two primer-binding sites (PBS) on either side of a unique 20-bp sequence (barcode N 20 ). (E) Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL4-3 insertion library shows excisions of the TN5MK transposon (1.4 kb) and upward shift of the supercoiled library versus the undigested library. Lane M, 2-log DNA ladder; 1, undigested insertion library; 2, I-SceI digested insertion library; 3, I-CeuI digested insertion library. (F) Location of TN5MK insertions for a subset of 7,559 transposon integrations (3,844 were unique). (G) Determination of enzymatic chewback rate for deletion size. The chewback rate was determined by treating a 4-kb fragment of linear dsDNA with RecJ f and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time and then halting enzymatic activity. Reactions were performed in triplicates. DNA concentrations were established by quantifying the fluorescence of PicoGreen in a plate reader in comparison to that of a dsDNA standard of known concentration. (H) Validation of deletion library. The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (∅) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1 to 4 are the pNL4-3 insertion library, and lanes 5 to 8 are the pNL4-3 deletion library. (I) pNL4-3 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e., right-tailed) histogram of deletion sizes in pNL4-3, with bins of 100 bp (shown in blue), is well-fit by a gamma distribution (green dashed line). (Inset) Number of deletions detected within each region of the HIV genome. (J) Deletion depth profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-3 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication, and bla, β-lactamase, the resistance marker.

    Article Snippet: Inserted transposons were excised by treatment with either meganuclease I-SceI or I-CeuI in CutSmart buffer (NEB).

    Techniques: In Vitro, Ligation, Plasmid Preparation, De-Phosphorylation Assay, Construct, Binding Assay, Sequencing, Activity Assay, Fluorescence, Concentration Assay, Marker

    Application of RanDeL-seq to map Zika virus (ZIKV) cis elements. (A) pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3′ end of the genome, a self-cleaving hepatitis delta virus ribozyme allows for creation of an authentic 3′ end posttranscription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. (B) Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“Ins.”). (+) and (−) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested completed deletion libraries (lanes 1 to 4) were run next to undigested insertion libraries (lanes 5 and 6). Insertion libraries (lanes 7 to 10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (lanes 11 to 14). (C) Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. (D) Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentages of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high-MOI screen and a wild-type ZIKV control (WT). RT-qPCR data were normalized to an MS2 RNA spike-in. (E) Deletion depth profile of intracellular RNA of 293T cotransfected with the wild-type ZIKV plasmid and the pooled deletion libraries. (F) Deletion depth profile of pMR766(+)L after passage 1. Only deletions in Pr to NS1 can be trans -complemented by wild-type ZIKV. (G) Final map of ZIKV cis - and trans -acting elements after passage 2. The two cis -acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans -acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).

    Journal: mBio

    Article Title: RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements

    doi: 10.1128/mBio.01724-20

    Figure Lengend Snippet: Application of RanDeL-seq to map Zika virus (ZIKV) cis elements. (A) pMR766(+), a Zika virus molecular clone. The MR766 Zika virus genome is encoded as a cDNA driven by the CMV IE2 promoter. At the 3′ end of the genome, a self-cleaving hepatitis delta virus ribozyme allows for creation of an authentic 3′ end posttranscription. An intron sequence is present within NS1 to allow maintenance in bacteria but is spliced out during transcription in host cells. (B) Restriction enzyme characterization of completed ZIKV deletion libraries compared to insertion libraries (“Ins.”). (+) and (−) designate the template ZIKV plasmid. “S” and “L” designate the chewback length for deletion libraries. Undigested completed deletion libraries (lanes 1 to 4) were run next to undigested insertion libraries (lanes 5 and 6). Insertion libraries (lanes 7 to 10) treated with I-SceI or I-CeuI to excise transposon (∼1.4 kb). Deletion libraries linearized by unique ZIKV cutter KpnI (lanes 11 to 14). (C) Deletion depth profile of the pMR766(+)L library. The ZIKV genome is well represented in the pMR766(+)L library, with some bias. Each base of the ZIKV genome is covered by several hundred different deletion mutants. (D) Detection and quantification of ZIKV barcode cassettes by RT-qPCR. Genomic percentages of barcoded mutants to total ZIKV genomes at each day in passage 1 of the high-MOI screen and a wild-type ZIKV control (WT). RT-qPCR data were normalized to an MS2 RNA spike-in. (E) Deletion depth profile of intracellular RNA of 293T cotransfected with the wild-type ZIKV plasmid and the pooled deletion libraries. (F) Deletion depth profile of pMR766(+)L after passage 1. Only deletions in Pr to NS1 can be trans -complemented by wild-type ZIKV. (G) Final map of ZIKV cis - and trans -acting elements after passage 2. The two cis -acting regions are highlighted in blue and do not tolerate deletion (i.e., must be present for efficient transmission to occur). The trans -acting region is highlighted in green and can be complemented in trans (i.e., if deleted, transmission occurs by complementation from wild-type virus).

    Article Snippet: Inserted transposons were excised by treatment with either meganuclease I-SceI or I-CeuI in CutSmart buffer (NEB).

    Techniques: Sequencing, Plasmid Preparation, Quantitative RT-PCR, Transmission Assay

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Journal: Genome Biology and Evolution

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    doi: 10.1093/gbe/evq056

    Figure Lengend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Article Snippet: All the 80 L. lactis strains examined gave six fragments when their genomic DNA was digested with I-Ceu I, indicating that the copy number of the rRNA genes is conserved in this species ( ).

    Techniques:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Journal: Genome Biology and Evolution

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    doi: 10.1093/gbe/evq056

    Figure Lengend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Article Snippet: All the 80 L. lactis strains examined gave six fragments when their genomic DNA was digested with I-Ceu I, indicating that the copy number of the rRNA genes is conserved in this species ( ).

    Techniques: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Journal: Genome Biology and Evolution

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    doi: 10.1093/gbe/evq056

    Figure Lengend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Article Snippet: All the 80 L. lactis strains examined gave six fragments when their genomic DNA was digested with I-Ceu I, indicating that the copy number of the rRNA genes is conserved in this species ( ).

    Techniques:

    Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A β-lactamases of S. fonticola ; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Molecular Characterization of a Carbapenem-Hydrolyzing Class A ?-Lactamase, SFC-1, from Serratia fonticola UTAD54

    doi: 10.1128/AAC.48.6.2321-2324.2004

    Figure Lengend Snippet: Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A β-lactamases of S. fonticola ; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.

    Article Snippet: DNA from S. fonticola UTAD54 embedded in agarose was digested with I-CeuI (New England Biolabs, Hertfordshire, United Kingdom), and the resulting fragments were separated on a CHEF-DRII apparatus (Bio-Rad, Richmond, Calif.) ( ).

    Techniques: Generated, Pulsed-Field Gel, Electrophoresis, Hybridization, Lambda DNA Preparation

    Localization of bla CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla CMY-2 (C), and int (D).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿

    doi: 10.1128/AAC.00111-10

    Figure Lengend Snippet: Localization of bla CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla CMY-2 (C), and int (D).

    Article Snippet: After digestion of whole-cell DNA with I-CeuI (New England Biolabs, Hertfordshire, United Kingdom), the resultant fragments were separated with a CHEF-Mapper apparatus (Bio-Rad, Hercules, CA) at 14°C, 6 V/cm, and a 120° switch angle, with a nonlinear switch time ramp of 5.3 to 49.9 s for 19.7 h. The sizes of the fragments were determined by comparison with a yeast chromosome pulsed-field gel electrophoresis marker (New England Biolabs).

    Techniques: Pulsed-Field Gel, Electrophoresis