lentiviral expression vector  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92

    Structured Review

    Thermo Fisher lentiviral expression vector
    Identification of EGFP-labeled <t>lentiviral</t> expression vector. (A-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-EGFR-miR. M, marker DL2000; lane 1, negative control; lanes 2–4, PCR product of recombinant vector (911 bp). (A-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-EGFR-miR. M, marker DL15000; lane 1, digestion product of double restriction endonuclease of Asc I and Pme I; lane 2, the recombinant plasmid pLenti6.3-EGFR-EGFR-miR. (B-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, negative control; lanes 2–6, PCR product of recombinant vector (911 bp). (B-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, digestion product of double restriction endonuclease of AscI and PmeI; lane 2, the recombinant plasmid pLenti6.3-EGFP-FGFR-miR. EGFP, enhanced green fluorescent protein; EGFR, epithelial growth factor receptor; miR, microRNA; PCR, polymerase chain reaction; FGFR, fibroblast growth factor receptor.
    Lentiviral Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 5428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentiviral expression vector/product/Thermo Fisher
    Average 92 stars, based on 5428 article reviews
    Price from $9.99 to $1999.99
    lentiviral expression vector - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes"

    Article Title: Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1685

    Identification of EGFP-labeled lentiviral expression vector. (A-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-EGFR-miR. M, marker DL2000; lane 1, negative control; lanes 2–4, PCR product of recombinant vector (911 bp). (A-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-EGFR-miR. M, marker DL15000; lane 1, digestion product of double restriction endonuclease of Asc I and Pme I; lane 2, the recombinant plasmid pLenti6.3-EGFR-EGFR-miR. (B-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, negative control; lanes 2–6, PCR product of recombinant vector (911 bp). (B-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, digestion product of double restriction endonuclease of AscI and PmeI; lane 2, the recombinant plasmid pLenti6.3-EGFP-FGFR-miR. EGFP, enhanced green fluorescent protein; EGFR, epithelial growth factor receptor; miR, microRNA; PCR, polymerase chain reaction; FGFR, fibroblast growth factor receptor.
    Figure Legend Snippet: Identification of EGFP-labeled lentiviral expression vector. (A-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-EGFR-miR. M, marker DL2000; lane 1, negative control; lanes 2–4, PCR product of recombinant vector (911 bp). (A-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-EGFR-miR. M, marker DL15000; lane 1, digestion product of double restriction endonuclease of Asc I and Pme I; lane 2, the recombinant plasmid pLenti6.3-EGFR-EGFR-miR. (B-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, negative control; lanes 2–6, PCR product of recombinant vector (911 bp). (B-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, digestion product of double restriction endonuclease of AscI and PmeI; lane 2, the recombinant plasmid pLenti6.3-EGFP-FGFR-miR. EGFP, enhanced green fluorescent protein; EGFR, epithelial growth factor receptor; miR, microRNA; PCR, polymerase chain reaction; FGFR, fibroblast growth factor receptor.

    Techniques Used: Labeling, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control, Recombinant

    2) Product Images from "Exploring potassium-dependent GTP hydrolysis in TEES family GTPases"

    Article Title: Exploring potassium-dependent GTP hydrolysis in TEES family GTPases

    Journal: FEBS Open Bio

    doi: 10.1016/j.fob.2012.07.008

    K + ion stabilizes the transition state. Nucleotide binding assays, employing fluorescent N-methyl-2’/3’-O-anthraniloyl nucleotides GDP (mGDP) were carried out to assay stabilization of the transition state mimic mGDP·AlF x in presence of monovalent ions KCl and NaCl. (A) The emission spectra, monitored at 380–600 nm, show that the presence of K + ions enhances the binding of the transition state analogue (mGDP·AlF x ) in full length YphC-WT (spectra 6), while Na + does not show a similar effect (spectra 5). (B) GD1-YphC shows a similar trend in enhancing the binding of the transition state analog only in the presence of K + , (C) while GD2-YphC does not display a similar trend. (D) Era too shows significant binding of mGDP·AlF x in presence of K + , but not with Na + .
    Figure Legend Snippet: K + ion stabilizes the transition state. Nucleotide binding assays, employing fluorescent N-methyl-2’/3’-O-anthraniloyl nucleotides GDP (mGDP) were carried out to assay stabilization of the transition state mimic mGDP·AlF x in presence of monovalent ions KCl and NaCl. (A) The emission spectra, monitored at 380–600 nm, show that the presence of K + ions enhances the binding of the transition state analogue (mGDP·AlF x ) in full length YphC-WT (spectra 6), while Na + does not show a similar effect (spectra 5). (B) GD1-YphC shows a similar trend in enhancing the binding of the transition state analog only in the presence of K + , (C) while GD2-YphC does not display a similar trend. (D) Era too shows significant binding of mGDP·AlF x in presence of K + , but not with Na + .

    Techniques Used: Binding Assay

    Mutating P-loop asparagines in YphC and Era, abolishes K + ion dependent stimulation of GTP hydrolysis and transition state stabilization. A conserved Asn in the P-loop, critical for coordinating the K + ion, was mutated to alanine in YphC and Era to create YphC-N13A (of GD1), YphC-N185A (of GD2) and Era-N18A mutants. GTP hydrolysis in presence of various ions (indicated in the insets) were measured using [α 32 P]-GTP. Autoradiograms show the hydrolysis of GTP to GDP, in presence of various salts (as indicated), by (A) YphC-N13A, (B) by YphC-N185A and (C) by Era-N18A. In all cases, a control devoid of protein was included. Fluorescent mGDP binding assays, as in Fig. 4 , were carried out for (D) YphC-N13A (E) YphC-N185A and (F) Era-N18A. Unlike with wild type proteins ( Fig. 4 ), in these, enhanced binding of the transition state mimic mGDP·AlF x in the presence of KCl is not observed for YphC-N13A and Era-N18A. This is in line with the observations that Yphc-GD1 and Era use a K + dependent stimulation of GTP hydrolysis.
    Figure Legend Snippet: Mutating P-loop asparagines in YphC and Era, abolishes K + ion dependent stimulation of GTP hydrolysis and transition state stabilization. A conserved Asn in the P-loop, critical for coordinating the K + ion, was mutated to alanine in YphC and Era to create YphC-N13A (of GD1), YphC-N185A (of GD2) and Era-N18A mutants. GTP hydrolysis in presence of various ions (indicated in the insets) were measured using [α 32 P]-GTP. Autoradiograms show the hydrolysis of GTP to GDP, in presence of various salts (as indicated), by (A) YphC-N13A, (B) by YphC-N185A and (C) by Era-N18A. In all cases, a control devoid of protein was included. Fluorescent mGDP binding assays, as in Fig. 4 , were carried out for (D) YphC-N13A (E) YphC-N185A and (F) Era-N18A. Unlike with wild type proteins ( Fig. 4 ), in these, enhanced binding of the transition state mimic mGDP·AlF x in the presence of KCl is not observed for YphC-N13A and Era-N18A. This is in line with the observations that Yphc-GD1 and Era use a K + dependent stimulation of GTP hydrolysis.

    Techniques Used: Binding Assay

    Potassium dependent GTPase activity in EngB, HflX and the two G domains GD1 and GD2 of YphC. Individual G domains, GD1-YphC and GD2-YphC were generated and used to determine the effect of K + and Na + ions on GTP hydrolysis by the two domains, like in Fig. 2 . (A) An autoradiogram showing the hydrolysis of GTP to GDP by GD1-YphC (left panel) and GD2-YphC-(right panel) in presence of 200 mM NaCl and 200 mM KCl, along with a control devoid of protein. Amount of GTP hydrolyzed in presence of salts was quantified (from the above autoradiograms) and represented as fold stimulation considering GTP hydrolysis in presence of Na + to be 1-fold. The experiments were repeated thrice and error bars are shown. GTP hydrolysis assays were conducted as above, for (B) EngB and (C) HflX.
    Figure Legend Snippet: Potassium dependent GTPase activity in EngB, HflX and the two G domains GD1 and GD2 of YphC. Individual G domains, GD1-YphC and GD2-YphC were generated and used to determine the effect of K + and Na + ions on GTP hydrolysis by the two domains, like in Fig. 2 . (A) An autoradiogram showing the hydrolysis of GTP to GDP by GD1-YphC (left panel) and GD2-YphC-(right panel) in presence of 200 mM NaCl and 200 mM KCl, along with a control devoid of protein. Amount of GTP hydrolyzed in presence of salts was quantified (from the above autoradiograms) and represented as fold stimulation considering GTP hydrolysis in presence of Na + to be 1-fold. The experiments were repeated thrice and error bars are shown. GTP hydrolysis assays were conducted as above, for (B) EngB and (C) HflX.

    Techniques Used: Activity Assay, Generated

    3) Product Images from "Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes"

    Article Title: Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-99

    (A) Green fluorescent protein (GFP) constructs evaluated for the promoter activity of p28-Omp genes 14 and 19 . The pPROBE-NT plasmids containing the promoterless GFP gene (2 and 3) and upstream sequences of genes 14 and 19 in front of the GFP gene (1 and 4, respectively) and a construct containing no promoter sequence were evaluated for GFP expression in E. coli . (B) LacZ constructs evaluated for the promoter activity of p28-Omp genes 14 and 19. The pBlue-TOPO vector containing promoterless lacZ gene (pBlue-TOPO) and upstream sequences of genes 14 and 19 inserted in forward (14-F and 19-F) and reverse orientations (14-R and 19-R) were evaluated for β-galactosidase activity in E. coli . Data are presented with SD values calculated from four independent experiments ( P ≤ 0.001).
    Figure Legend Snippet: (A) Green fluorescent protein (GFP) constructs evaluated for the promoter activity of p28-Omp genes 14 and 19 . The pPROBE-NT plasmids containing the promoterless GFP gene (2 and 3) and upstream sequences of genes 14 and 19 in front of the GFP gene (1 and 4, respectively) and a construct containing no promoter sequence were evaluated for GFP expression in E. coli . (B) LacZ constructs evaluated for the promoter activity of p28-Omp genes 14 and 19. The pBlue-TOPO vector containing promoterless lacZ gene (pBlue-TOPO) and upstream sequences of genes 14 and 19 inserted in forward (14-F and 19-F) and reverse orientations (14-R and 19-R) were evaluated for β-galactosidase activity in E. coli . Data are presented with SD values calculated from four independent experiments ( P ≤ 0.001).

    Techniques Used: Construct, Activity Assay, Sequencing, Expressing, Plasmid Preparation

    4) Product Images from "STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells"

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-67

    (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents
    Figure Legend Snippet: (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents "%" distribution.

    Techniques Used: Chromatin Immunoprecipitation, Clone Assay, Sonication, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Immunoprecipitation, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Genomic Sequencing

    5) Product Images from "Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)"

    Article Title: Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-42-3

    RFLP analysis of pBR- and pBRf-derived viruses and characterization of mini-F loss in pBR-derived viruses . A. BAC DNA or viral DNA prepared from infected Vero cells was digested with XhoI and separated by 0.8% agarose gel electrophoresis. The lanes contain the DNA's from BAC DNA (pBRf, pBR), viruses reconstituted from cloned DNA (vBRf, vBR) or parental CPXV.BR DNA (BR). Changes of the restriction pattern due to the presence and therefore disruption of TK of absence of mini-F sequences are marked with boxes (see text). B. Southern blot analysis after transfer of the gel shown in (A) and using a DIG labeled TK probe. The changes seen with a DIG labeled TK probe show the expected changes due to the disruption of TK with mini-F sequences (pBRf, vBRf), the introduction of an inverse TK sequence (pBR), and the loss of any bacterial sequences in vBR (see text). Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. C. Development of GFP fluorescence in plaques following removal of mini-F sequences. Initially, all plaques exhibit GFP fluorescence (plaque in left panels), which is then partially lost (plaque in middle panels) or completely absent (plaque in right panels). Identical plaques were photographed under fluorescent light (upper panels) or using phase contrast (lower panels) with a Zeiss Axiovert microscope and a CCD camera (Zeiss).
    Figure Legend Snippet: RFLP analysis of pBR- and pBRf-derived viruses and characterization of mini-F loss in pBR-derived viruses . A. BAC DNA or viral DNA prepared from infected Vero cells was digested with XhoI and separated by 0.8% agarose gel electrophoresis. The lanes contain the DNA's from BAC DNA (pBRf, pBR), viruses reconstituted from cloned DNA (vBRf, vBR) or parental CPXV.BR DNA (BR). Changes of the restriction pattern due to the presence and therefore disruption of TK of absence of mini-F sequences are marked with boxes (see text). B. Southern blot analysis after transfer of the gel shown in (A) and using a DIG labeled TK probe. The changes seen with a DIG labeled TK probe show the expected changes due to the disruption of TK with mini-F sequences (pBRf, vBRf), the introduction of an inverse TK sequence (pBR), and the loss of any bacterial sequences in vBR (see text). Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. C. Development of GFP fluorescence in plaques following removal of mini-F sequences. Initially, all plaques exhibit GFP fluorescence (plaque in left panels), which is then partially lost (plaque in middle panels) or completely absent (plaque in right panels). Identical plaques were photographed under fluorescent light (upper panels) or using phase contrast (lower panels) with a Zeiss Axiovert microscope and a CCD camera (Zeiss).

    Techniques Used: Derivative Assay, BAC Assay, Infection, Agarose Gel Electrophoresis, Clone Assay, Southern Blot, Labeling, Sequencing, Molecular Weight, Marker, Fluorescence, Microscopy

    Restriction fragment analysis of full-length BAC clone pBRf . A. BAC DNA was isolated and digested with seven different enzymes (HindIII, KpnI, PstI, SacI, SphI, StuI, XhoI) and separated on a 0.8% agarose gel. Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. B. Patterns corresponded exactly to the predictions based on the complete CPXV.BR genome (#NC_003663) as shown by in silico digests using VectorNTI.
    Figure Legend Snippet: Restriction fragment analysis of full-length BAC clone pBRf . A. BAC DNA was isolated and digested with seven different enzymes (HindIII, KpnI, PstI, SacI, SphI, StuI, XhoI) and separated on a 0.8% agarose gel. Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. B. Patterns corresponded exactly to the predictions based on the complete CPXV.BR genome (#NC_003663) as shown by in silico digests using VectorNTI.

    Techniques Used: BAC Assay, Isolation, Agarose Gel Electrophoresis, Molecular Weight, Marker, In Silico

    Schematic presentation of rationale for cloning of cowpox virus infectious clone . A. Shown is a schematic of the full-length CPXV.BR genome and the TK locus in greater detail. In a first step, the viral TK was amplified by PCR, cloned into the pCRII vector, which was then used for an inverse PCR to introduce an Fse I restriction site into TK. Mini-F vector sequences containing gfp under the control of the late 4B promoter were finally inserted into the singular Fse I site to obtain transfer plasmid #7. B. Shown are the constructs and the strategy used to obtain recombinant virus BR.TK- and the full-length CPXV.BR BAC pBRf. Recombinant BR.TK- was used to infect Vero cells. Infection resulted in the formation of replication intermediates, concatemers, some of which were circularized. Viral DNA extracted from cells infected with BR.TK- was electroporated into E. coli DH10B giving rise to pBRf. The full-length CPXV BAC clone was ultimately transfected into Vero cells using FWPV as a helper. Scale bars indicate the sizes of the molecules. Abbreviations: ITR, inverted terminal repeats; cat , chloramphenicol resistance gene; gfp , green fluorescent protein gene: loxP , loxP sites; H,
    Figure Legend Snippet: Schematic presentation of rationale for cloning of cowpox virus infectious clone . A. Shown is a schematic of the full-length CPXV.BR genome and the TK locus in greater detail. In a first step, the viral TK was amplified by PCR, cloned into the pCRII vector, which was then used for an inverse PCR to introduce an Fse I restriction site into TK. Mini-F vector sequences containing gfp under the control of the late 4B promoter were finally inserted into the singular Fse I site to obtain transfer plasmid #7. B. Shown are the constructs and the strategy used to obtain recombinant virus BR.TK- and the full-length CPXV.BR BAC pBRf. Recombinant BR.TK- was used to infect Vero cells. Infection resulted in the formation of replication intermediates, concatemers, some of which were circularized. Viral DNA extracted from cells infected with BR.TK- was electroporated into E. coli DH10B giving rise to pBRf. The full-length CPXV BAC clone was ultimately transfected into Vero cells using FWPV as a helper. Scale bars indicate the sizes of the molecules. Abbreviations: ITR, inverted terminal repeats; cat , chloramphenicol resistance gene; gfp , green fluorescent protein gene: loxP , loxP sites; H, "head", T, "tail" orientation of the ITR present in the replicative intermediates.

    Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Inverse PCR, Introduce, Construct, Recombinant, BAC Assay, Infection, Transfection

    6) Product Images from "Limited functional conservation of a global regulator among related bacterial genera: Lrp in Escherichia, Proteus and Vibrio"

    Article Title: Limited functional conservation of a global regulator among related bacterial genera: Lrp in Escherichia, Proteus and Vibrio

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-60

    Genome-wide comparison of transcriptional effects of three Lrp orthologs. The Venn diagram shows subsets of E. coli genes that were differentially regulated in response to Lrp orthologs from the indicated species (but not to the vector control). Gene expression was assessed by two-color microarray analysis as described in Methods. The pie chart represents the relative distribution of genes significantly responsive to the E. coli Lrp that are also significantly responsive to the other Lrp orthologs. Details of the statistical analysis of these data are in Methods, and the gene-specific results are available [see Additional file 2 ].
    Figure Legend Snippet: Genome-wide comparison of transcriptional effects of three Lrp orthologs. The Venn diagram shows subsets of E. coli genes that were differentially regulated in response to Lrp orthologs from the indicated species (but not to the vector control). Gene expression was assessed by two-color microarray analysis as described in Methods. The pie chart represents the relative distribution of genes significantly responsive to the E. coli Lrp that are also significantly responsive to the other Lrp orthologs. Details of the statistical analysis of these data are in Methods, and the gene-specific results are available [see Additional file 2 ].

    Techniques Used: Genome Wide, Plasmid Preparation, Expressing, Microarray

    Regulation of selected target genes by heterologous Lrp proteins. E. coli strains, all carrying lrp-Tn 10 and Δ lac , were transformed with plasmids carrying various lrp alleles (or vector control). Transformants were grown in unsupplemented MOPS glucose medium. A . Western blot analysis of Lrp accumulation (Eco, E. coli Lrp; Pmi, P. mirabilis Lrp; Vch, V. cholerae Lrp; pCC1, vector control) using polyclonal antiserum raised against E. coli Lrp. The arrow indicates the direction of electrophoresis. B-D . P lrp-lacZ (B), P gltB-lacZ (C) and P livK-lacZ (D) activity were measured via ONPG hydrolysis, and plotted vs . culture density to ensure that the cultures were in balanced growth. The Lrp orthologs used are from P. mirabilis (triangles) and V. cholerae (squares), as well as the E. coli positive control (circles) and the vector control (diamonds). E-G . Isoleucine, Leucine and Valine was added to the medium (
    Figure Legend Snippet: Regulation of selected target genes by heterologous Lrp proteins. E. coli strains, all carrying lrp-Tn 10 and Δ lac , were transformed with plasmids carrying various lrp alleles (or vector control). Transformants were grown in unsupplemented MOPS glucose medium. A . Western blot analysis of Lrp accumulation (Eco, E. coli Lrp; Pmi, P. mirabilis Lrp; Vch, V. cholerae Lrp; pCC1, vector control) using polyclonal antiserum raised against E. coli Lrp. The arrow indicates the direction of electrophoresis. B-D . P lrp-lacZ (B), P gltB-lacZ (C) and P livK-lacZ (D) activity were measured via ONPG hydrolysis, and plotted vs . culture density to ensure that the cultures were in balanced growth. The Lrp orthologs used are from P. mirabilis (triangles) and V. cholerae (squares), as well as the E. coli positive control (circles) and the vector control (diamonds). E-G . Isoleucine, Leucine and Valine was added to the medium ("+Leu") for experiments depicted in the lower panels: Plrp-lacZ (E), PgltB-lacZ (F) and PlivK-lacZ (G). The correlation coefficients for the least-squares fits to the data were all at least 0.97.

    Techniques Used: Transformation Assay, Plasmid Preparation, Western Blot, Electrophoresis, Activity Assay, Positive Control

    Sequences of selected Lrp proteins. Lrp proteins from various bacterial species were aligned; species used in this study are in bold ( Escherichia coli , Proteus mirabilis , and Vibrio cholerae ). A more complete list of Lrp orthologs and paralogs can be found online [140]. The gray-shaded regions indicate N- and C-terminal sequences that are conserved among enterobacterial Lrp orthologs, and the black-shaded regions indicate substitutions relative to E. coli . The boxed regions indicate the DNA-binding helix-turn-helix motif (top portion, under cartoon representation), and the leucine-binding sites (lower portion of sequence); all boxed regions are completely conserved among the species used in this study. For references, see main text. Other Lrp orthologs shown came from (in order shown): Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae , Yersinia pestis , Haemophilus influenzae Rd, Actinobacillus pleuropneumoniae , Pasteurella multocida , and Pseudomonas aeruginosa .
    Figure Legend Snippet: Sequences of selected Lrp proteins. Lrp proteins from various bacterial species were aligned; species used in this study are in bold ( Escherichia coli , Proteus mirabilis , and Vibrio cholerae ). A more complete list of Lrp orthologs and paralogs can be found online [140]. The gray-shaded regions indicate N- and C-terminal sequences that are conserved among enterobacterial Lrp orthologs, and the black-shaded regions indicate substitutions relative to E. coli . The boxed regions indicate the DNA-binding helix-turn-helix motif (top portion, under cartoon representation), and the leucine-binding sites (lower portion of sequence); all boxed regions are completely conserved among the species used in this study. For references, see main text. Other Lrp orthologs shown came from (in order shown): Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae , Yersinia pestis , Haemophilus influenzae Rd, Actinobacillus pleuropneumoniae , Pasteurella multocida , and Pseudomonas aeruginosa .

    Techniques Used: Binding Assay, Sequencing

    Regulation of orthologous target genes in native backgrounds. A-C: Sequences upstream of adhE , gltB and lrp orthologs. In each case, the sequence ends with the initiation codon. Lrp-binding sites and the transcriptional +1 position are known for E. coli K -12 [112]. Demonstrated Lrp binding sites are in underlined lowercase italics , and the -35 and -10 sequences inferred from the known +1 position (for E. coli ) are boxed. Putative binding sites, predicted by the PRODORIC virtual footprinter [102] are shaded, and the match scores for predicted sites are shown to the right. For E. coli P gltB , one of the predicted sites overlaps an actual site, and gives a particularly high match score, though an overlapping actual site in P lrp does not. V. cholerae has two nearly-tandem copies of the gltBD operon on chromosome I. The 5'-most gltB isozyme (
    Figure Legend Snippet: Regulation of orthologous target genes in native backgrounds. A-C: Sequences upstream of adhE , gltB and lrp orthologs. In each case, the sequence ends with the initiation codon. Lrp-binding sites and the transcriptional +1 position are known for E. coli K -12 [112]. Demonstrated Lrp binding sites are in underlined lowercase italics , and the -35 and -10 sequences inferred from the known +1 position (for E. coli ) are boxed. Putative binding sites, predicted by the PRODORIC virtual footprinter [102] are shaded, and the match scores for predicted sites are shown to the right. For E. coli P gltB , one of the predicted sites overlaps an actual site, and gives a particularly high match score, though an overlapping actual site in P lrp does not. V. cholerae has two nearly-tandem copies of the gltBD operon on chromosome I. The 5'-most gltB isozyme ("Vch1", locus tag Vc2373) is 43% identical to Eco gltB , while the 3'-most isozyme ("Vch2", Vc2376) is 73% identical to Eco gltB in amino acid sequence. D-E: Samples were isolated at an OD 600 nm of 0.3 (log), as well as 1 h after linear growth stopped (stationary), from E. coli, P. mirabilis and V. cholerae wild-type and lrp cultures growing in MOPS defined rich medium. QRT-PCR was used to determine the relative levels of adhE, gltB and recA messages, with recA serving to provide a Lrp-independent baseline. The experiment was performed in triplicate and the level of message was determined using the standard curve method and normalization to recA . D – adhE . E – gltB . For each plot filled symbols represent log phase levels and open symbols represent stationary phase levels. The symbol shapes indicate the species: P. mirabilis (triangles) E. coli (circles) or V. cholerae (Vc2373, squares). The line indicates the position for data if no effect of Lrp is seen (ratio of 1); points above the line are consistent with repression, while those below the line are consistent with activation by Lrp. The dotted lines show, to facilitate comparison, the borders of a twofold effect.

    Techniques Used: Sequencing, Binding Assay, Isolation, Quantitative RT-PCR, Activation Assay

    7) Product Images from "Limited functional conservation of a global regulator among related bacterial genera: Lrp in Escherichia, Proteus and Vibrio"

    Article Title: Limited functional conservation of a global regulator among related bacterial genera: Lrp in Escherichia, Proteus and Vibrio

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-60

    Regulation of orthologous target genes in native backgrounds. A-C: Sequences upstream of adhE , gltB and lrp orthologs. In each case, the sequence ends with the initiation codon. Lrp-binding sites and the transcriptional +1 position are known for E. coli K -12 [112]. Demonstrated Lrp binding sites are in underlined lowercase italics , and the -35 and -10 sequences inferred from the known +1 position (for E. coli ) are boxed. Putative binding sites, predicted by the PRODORIC virtual footprinter [102] are shaded, and the match scores for predicted sites are shown to the right. For E. coli P gltB , one of the predicted sites overlaps an actual site, and gives a particularly high match score, though an overlapping actual site in P lrp does not. V. cholerae has two nearly-tandem copies of the gltBD operon on chromosome I. The 5'-most gltB isozyme (
    Figure Legend Snippet: Regulation of orthologous target genes in native backgrounds. A-C: Sequences upstream of adhE , gltB and lrp orthologs. In each case, the sequence ends with the initiation codon. Lrp-binding sites and the transcriptional +1 position are known for E. coli K -12 [112]. Demonstrated Lrp binding sites are in underlined lowercase italics , and the -35 and -10 sequences inferred from the known +1 position (for E. coli ) are boxed. Putative binding sites, predicted by the PRODORIC virtual footprinter [102] are shaded, and the match scores for predicted sites are shown to the right. For E. coli P gltB , one of the predicted sites overlaps an actual site, and gives a particularly high match score, though an overlapping actual site in P lrp does not. V. cholerae has two nearly-tandem copies of the gltBD operon on chromosome I. The 5'-most gltB isozyme ("Vch1", locus tag Vc2373) is 43% identical to Eco gltB , while the 3'-most isozyme ("Vch2", Vc2376) is 73% identical to Eco gltB in amino acid sequence. D-E: Samples were isolated at an OD 600 nm of 0.3 (log), as well as 1 h after linear growth stopped (stationary), from E. coli, P. mirabilis and V. cholerae wild-type and lrp cultures growing in MOPS defined rich medium. QRT-PCR was used to determine the relative levels of adhE, gltB and recA messages, with recA serving to provide a Lrp-independent baseline. The experiment was performed in triplicate and the level of message was determined using the standard curve method and normalization to recA . D – adhE . E – gltB . For each plot filled symbols represent log phase levels and open symbols represent stationary phase levels. The symbol shapes indicate the species: P. mirabilis (triangles) E. coli (circles) or V. cholerae (Vc2373, squares). The line indicates the position for data if no effect of Lrp is seen (ratio of 1); points above the line are consistent with repression, while those below the line are consistent with activation by Lrp. The dotted lines show, to facilitate comparison, the borders of a twofold effect.

    Techniques Used: Sequencing, Binding Assay, Isolation, Quantitative RT-PCR, Activation Assay

    8) Product Images from "Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes"

    Article Title: Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002169

    Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or Cry21Aa2 (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .
    Figure Legend Snippet: Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or Cry21Aa2 (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .

    Techniques Used: Plasmid Preparation, Expressing

    Frequency of BT-679 toxin genes cry21Aa2 and cry35Aa4 among the evolved replicate populations. The different shades of blue indicate alternative combinations of toxin genes present, as indicated. The toxin genes were all restricted to evolved clones of the BT-679 background (i.e., horizontal transfer was not detected). The top two rows refer to the coevolved, the middle two rows to one-sided adapted, and the bottom two rows to the control evolved replicate populations. Replicate populations are given along the horizontal axis. Data is shown for both transfer 12 and 20 and a total of 55 replicate populations. Crosses indicate extinction of replicates and
    Figure Legend Snippet: Frequency of BT-679 toxin genes cry21Aa2 and cry35Aa4 among the evolved replicate populations. The different shades of blue indicate alternative combinations of toxin genes present, as indicated. The toxin genes were all restricted to evolved clones of the BT-679 background (i.e., horizontal transfer was not detected). The top two rows refer to the coevolved, the middle two rows to one-sided adapted, and the bottom two rows to the control evolved replicate populations. Replicate populations are given along the horizontal axis. Data is shown for both transfer 12 and 20 and a total of 55 replicate populations. Crosses indicate extinction of replicates and "miss" that genetic material for the population was unavailable. The original data is shown in S4 Data .

    Techniques Used: Clone Assay

    9) Product Images from "The Role of TcdB and TccC Subunits in Secretion of the Photorhabdus Tcd Toxin Complex"

    Article Title: The Role of TcdB and TccC Subunits in Secretion of the Photorhabdus Tcd Toxin Complex

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003644

    The N-terminal 361 amino acids of TcdB1 are not essential for toxicity. Mean larval weight (MLW) gain of cohorts of M. sexta neonates (n = 10) fed 100 µl of mixtures of sonicated cell extracts (L) and culture supernatants (S). Arabinose inducible expression constructs tested include the pBAD30 vector only, “V”, the tcdA1 only construct, “pA” ( figure 4 ) and the BC5 and BΔC constructs ( figure 6 ). Sample mixes were as either (i) 50 µl lysed pA sample complemented with 950 µl induced supernatant (left panel) or (ii) 50 µl lysed pA sample complemented with 50 µl lysed test sample and diluted with 900 µl of PBS. Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. A key using the data column fill pattern is given above the graph to assist in the interpretation of the predicted TC subunit contents of the test samples. The indicators * and ** are discussed in text. Two sample t -test comparisons were used to confirm statistical significance in mean weight differences at 99% confidence between the following samples discussed in the text; [pA(L)+BΔC(S)] vs. [pA(L)+BC5(S)]: t = 26.68, P
    Figure Legend Snippet: The N-terminal 361 amino acids of TcdB1 are not essential for toxicity. Mean larval weight (MLW) gain of cohorts of M. sexta neonates (n = 10) fed 100 µl of mixtures of sonicated cell extracts (L) and culture supernatants (S). Arabinose inducible expression constructs tested include the pBAD30 vector only, “V”, the tcdA1 only construct, “pA” ( figure 4 ) and the BC5 and BΔC constructs ( figure 6 ). Sample mixes were as either (i) 50 µl lysed pA sample complemented with 950 µl induced supernatant (left panel) or (ii) 50 µl lysed pA sample complemented with 50 µl lysed test sample and diluted with 900 µl of PBS. Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. A key using the data column fill pattern is given above the graph to assist in the interpretation of the predicted TC subunit contents of the test samples. The indicators * and ** are discussed in text. Two sample t -test comparisons were used to confirm statistical significance in mean weight differences at 99% confidence between the following samples discussed in the text; [pA(L)+BΔC(S)] vs. [pA(L)+BC5(S)]: t = 26.68, P

    Techniques Used: Sonication, Expressing, Construct, Plasmid Preparation

    Cosmid complementation bioassay showing the B+C sub-complex secretion is independent of the A-subunit. Mean larval weight (MLW) gain of cohorts of M. sexta neonates (n = 10) fed mixtures of sonicated cell extracts (L) and culture supernatants (S), at dilutions of 1∶5, 1∶10 or 1∶20. Samples were mixed at a ratio of 50 µl of lysate (L) to 950 µl of supernatants (S). Samples were taken from the cosmid mutant AKO ( figure 1 ) and pBAD30 expression constructs containing either nothing, “V”, or the tcdA1 gene only, “pA” [22] . Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. A key using the data column fill pattern is given above the graph to assist in the interpretation of the predicted TC subunit contents of the test samples. The indicators * and ** are discussed in text. Two sample t -test comparisons were used to confirm statistical significance in mean weight differences at 99% confidence between the following samples discussed in the text; [AKO(S)+V(L) 1∶5] vs. [AKO(S)+pA(L) 1∶5]: t = 47.75, P
    Figure Legend Snippet: Cosmid complementation bioassay showing the B+C sub-complex secretion is independent of the A-subunit. Mean larval weight (MLW) gain of cohorts of M. sexta neonates (n = 10) fed mixtures of sonicated cell extracts (L) and culture supernatants (S), at dilutions of 1∶5, 1∶10 or 1∶20. Samples were mixed at a ratio of 50 µl of lysate (L) to 950 µl of supernatants (S). Samples were taken from the cosmid mutant AKO ( figure 1 ) and pBAD30 expression constructs containing either nothing, “V”, or the tcdA1 gene only, “pA” [22] . Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. A key using the data column fill pattern is given above the graph to assist in the interpretation of the predicted TC subunit contents of the test samples. The indicators * and ** are discussed in text. Two sample t -test comparisons were used to confirm statistical significance in mean weight differences at 99% confidence between the following samples discussed in the text; [AKO(S)+V(L) 1∶5] vs. [AKO(S)+pA(L) 1∶5]: t = 47.75, P

    Techniques Used: Sonication, Mutagenesis, Expressing, Construct

    The cosmid model for heterologous TC synthesis and secretion in E. coli . ( A ) Map of cosmid (c1AH10) containing a portion of the P. luminescens W14 tcd pathogenicity island (bounded by dotted lines). Both strands are shown and the ORFs encoded on these are show as boxes. TC genes are shaded in grey and the pdl1 release lipase gene is hatched [27] . Inverted triangles show the location of transposon insertion mutants. The AKO, BKO and CKO insertion points are at nucleotides 271, 730 and 562 respectively, with respect to the first nucleotides of the ORFs. Note “H3” represents a cosmid clone in which the transposon has inserted into the vector backbone outside of the insert and therefore contains the full intact functional locus. ( B ) Western blot analysis of whole cells (200 µl culture pellet) and concentrated supernatants (1.2 ml) from the cosmid model of various transposon insertion mutant clones. Supernatants are from 2 day old cultures grown at 28°C in LB medium. Antibodies used were raised against the C-terminus of the B-subunit, TcdB1. Two independent B1 KO clones where tested (bar). An antibody directed against β-lactamase (Anti-Bla) is used as a loading control for the quality of the soluble and membrane fractions in this and subsequent blots. Note that knock out (KO) of the C-subunit tccC5 gene (CKO) prevents secretion of the B-subunit (*), while KO of the A-subunit tcdA1 gene (AKO) does not prevent B-subunit secretion (arrow). Bioassays of these same samples can be seen in Figure S1 .
    Figure Legend Snippet: The cosmid model for heterologous TC synthesis and secretion in E. coli . ( A ) Map of cosmid (c1AH10) containing a portion of the P. luminescens W14 tcd pathogenicity island (bounded by dotted lines). Both strands are shown and the ORFs encoded on these are show as boxes. TC genes are shaded in grey and the pdl1 release lipase gene is hatched [27] . Inverted triangles show the location of transposon insertion mutants. The AKO, BKO and CKO insertion points are at nucleotides 271, 730 and 562 respectively, with respect to the first nucleotides of the ORFs. Note “H3” represents a cosmid clone in which the transposon has inserted into the vector backbone outside of the insert and therefore contains the full intact functional locus. ( B ) Western blot analysis of whole cells (200 µl culture pellet) and concentrated supernatants (1.2 ml) from the cosmid model of various transposon insertion mutant clones. Supernatants are from 2 day old cultures grown at 28°C in LB medium. Antibodies used were raised against the C-terminus of the B-subunit, TcdB1. Two independent B1 KO clones where tested (bar). An antibody directed against β-lactamase (Anti-Bla) is used as a loading control for the quality of the soluble and membrane fractions in this and subsequent blots. Note that knock out (KO) of the C-subunit tccC5 gene (CKO) prevents secretion of the B-subunit (*), while KO of the A-subunit tcdA1 gene (AKO) does not prevent B-subunit secretion (arrow). Bioassays of these same samples can be seen in Figure S1 .

    Techniques Used: Plasmid Preparation, Functional Assay, Western Blot, Mutagenesis, Clone Assay, Knock-Out

    10) Product Images from "Medium-chain acyl-CoA dehydrogenase deficiency associated with a novel splice mutation in the ACADM gene missed by newborn screening"

    Article Title: Medium-chain acyl-CoA dehydrogenase deficiency associated with a novel splice mutation in the ACADM gene missed by newborn screening

    Journal: BMC Medical Genetics

    doi: 10.1186/s12881-015-0199-5

    A novel mutation in the ACADM gene results in aberrant splicing. Gel documentation visualizes the PCR product of a region of 323 bp from exon 7 to 9 of the ACADM gene in cDNA of control granulocytes (CTL GRAN) as well as of monocytes (MNC) and granulocytes (GRAN) of patient 1. Arrows point to the additional PCR products that were detected using cDNA of the patient’s granulocytes and monocytes. Analysis of the two additional PCR products in the gel eluates of the patient’s samples revealed missplicing resulting in either a partial loss of exon 8 (fragment of 294 bp, upper arrowhead) or complete skipping of exon 8 (fragment of 215 bp, lower arrowhead)
    Figure Legend Snippet: A novel mutation in the ACADM gene results in aberrant splicing. Gel documentation visualizes the PCR product of a region of 323 bp from exon 7 to 9 of the ACADM gene in cDNA of control granulocytes (CTL GRAN) as well as of monocytes (MNC) and granulocytes (GRAN) of patient 1. Arrows point to the additional PCR products that were detected using cDNA of the patient’s granulocytes and monocytes. Analysis of the two additional PCR products in the gel eluates of the patient’s samples revealed missplicing resulting in either a partial loss of exon 8 (fragment of 294 bp, upper arrowhead) or complete skipping of exon 8 (fragment of 215 bp, lower arrowhead)

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, CTL Assay

    11) Product Images from "Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae"

    Article Title: Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae

    Journal: SpringerPlus

    doi: 10.1186/s40064-016-1876-3

    Analysis of the process for FtsZ expression and purification by SDS-PAGE ( a ) and western blotting analysis of the purified FtsZ ( b ). The FtsZ was expressed in E. coli BL21 (DE3)/pET22b- ftsZ induced by 0.6 mM IPTG at 37 °C for 3 h. Lane M 1 and M 2 protein Marker; lane 1 purified FtsZ via Ni-NTA agarose column; lane 2 supernatant after ultrasonication under induced conditions; lane 3 total protein under induced conditions; lane 4 supernatant of broth under induced conditions; lane 5 supernatant of lysed E. coli BL21 cells containing pET-22b- ftsZ ; lane 6 purified FtsZ via Ni-NTA agarose column. The migration difference of the 44.3 kDa protein between ( a ) and ( b ) is due to gel electrophoresis with different pulse time
    Figure Legend Snippet: Analysis of the process for FtsZ expression and purification by SDS-PAGE ( a ) and western blotting analysis of the purified FtsZ ( b ). The FtsZ was expressed in E. coli BL21 (DE3)/pET22b- ftsZ induced by 0.6 mM IPTG at 37 °C for 3 h. Lane M 1 and M 2 protein Marker; lane 1 purified FtsZ via Ni-NTA agarose column; lane 2 supernatant after ultrasonication under induced conditions; lane 3 total protein under induced conditions; lane 4 supernatant of broth under induced conditions; lane 5 supernatant of lysed E. coli BL21 cells containing pET-22b- ftsZ ; lane 6 purified FtsZ via Ni-NTA agarose column. The migration difference of the 44.3 kDa protein between ( a ) and ( b ) is due to gel electrophoresis with different pulse time

    Techniques Used: Expressing, Purification, SDS Page, Western Blot, Marker, Positron Emission Tomography, Migration, Nucleic Acid Electrophoresis

    12) Product Images from "Lnc-CC3 increases metastasis in cervical cancer by increasing Slug expression"

    Article Title: Lnc-CC3 increases metastasis in cervical cancer by increasing Slug expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9519

    Lnc-CC3 enhanced the lung colonization capacity of SiHa cells ( A ) Representative images of nude mouse lungs; arrows indicate clusters of nodules on the lung surface. ( B ) Metastatic tumor nodules on the lung surface were counted under stereomicroscope. Each group contained 6 nude mice. ( C ) Hematoxylin-eosin staining of lung tumor nodules. Photographed at 100× magnification, scale bar = 400 μm. ( D ) Neoplasms were found in all pathological sections from lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SEM of independent experiments ( n = 6), * p
    Figure Legend Snippet: Lnc-CC3 enhanced the lung colonization capacity of SiHa cells ( A ) Representative images of nude mouse lungs; arrows indicate clusters of nodules on the lung surface. ( B ) Metastatic tumor nodules on the lung surface were counted under stereomicroscope. Each group contained 6 nude mice. ( C ) Hematoxylin-eosin staining of lung tumor nodules. Photographed at 100× magnification, scale bar = 400 μm. ( D ) Neoplasms were found in all pathological sections from lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SEM of independent experiments ( n = 6), * p

    Techniques Used: Mouse Assay, Staining

    lnc-CC3 full length clone and sequence analysis ( A ) Electrophoresis of the RACE product. ( B ) The full sequence of lnc-CC3. ( C ) The chromosomal location of lnc-CC3 was determined using NCBI MapViewer; the red label indicates its position. ( D ) Northern blot results for lnc-CC3. ( E ) In situ hybridization of lnc-CC3 in SiHa cells; the photograph was taken at 200× magnification. ( F ) Putative proteins encoded by lnc-CC3 as predicted by ORF Finder; predicted proteins were subject to Blastp. ( G ) Conservation analysis of lnc-CC3 was conducted using Clustal Omega; repeat sequence analysis was conducted using RepeatMasker.
    Figure Legend Snippet: lnc-CC3 full length clone and sequence analysis ( A ) Electrophoresis of the RACE product. ( B ) The full sequence of lnc-CC3. ( C ) The chromosomal location of lnc-CC3 was determined using NCBI MapViewer; the red label indicates its position. ( D ) Northern blot results for lnc-CC3. ( E ) In situ hybridization of lnc-CC3 in SiHa cells; the photograph was taken at 200× magnification. ( F ) Putative proteins encoded by lnc-CC3 as predicted by ORF Finder; predicted proteins were subject to Blastp. ( G ) Conservation analysis of lnc-CC3 was conducted using Clustal Omega; repeat sequence analysis was conducted using RepeatMasker.

    Techniques Used: Sequencing, Electrophoresis, Northern Blot, In Situ Hybridization

    Lnc-CC3 over-expression increased migration and invasion in SiHa cells in vitro ( A ) In situ hybridization results for the cervical tissue microarray revealed that lnc-CC3 is mainly localized in the cytoplasm. Normal, normal cervical tissue; CC _ Negative, cervical cancer negative; CC _ Positive, cervical cancer positive. 200× magnification, scale bar = 200 μm. ( B ) Lnc-CC3 levels were elevated more frequently in stage III cervical cancer. Normal, normal cervical tissue; I, II, III represent the different cervical cancer stages. Statistical significance was determined using the χ 2 test, p = 0.003. ( C ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( D ) Cell proliferation curve for SiHa cells determined using the CCK-8 assay ( n = 3). ( E ) Cell cycle distribution of SiHa cells was determined by flow cytometry ( n = 3). ( F ) Colony formation assay results for SiHa cells ( n = 3). ( G ) Cell migration capacity was determined using a wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification at the same location in each well. ( H ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Untransfected and pcDNA3.1 (+) plasmid transfected SiHa cell lines were used as controls; lnc-CC3 indicates lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SD of independent experiments, * p
    Figure Legend Snippet: Lnc-CC3 over-expression increased migration and invasion in SiHa cells in vitro ( A ) In situ hybridization results for the cervical tissue microarray revealed that lnc-CC3 is mainly localized in the cytoplasm. Normal, normal cervical tissue; CC _ Negative, cervical cancer negative; CC _ Positive, cervical cancer positive. 200× magnification, scale bar = 200 μm. ( B ) Lnc-CC3 levels were elevated more frequently in stage III cervical cancer. Normal, normal cervical tissue; I, II, III represent the different cervical cancer stages. Statistical significance was determined using the χ 2 test, p = 0.003. ( C ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( D ) Cell proliferation curve for SiHa cells determined using the CCK-8 assay ( n = 3). ( E ) Cell cycle distribution of SiHa cells was determined by flow cytometry ( n = 3). ( F ) Colony formation assay results for SiHa cells ( n = 3). ( G ) Cell migration capacity was determined using a wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification at the same location in each well. ( H ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Untransfected and pcDNA3.1 (+) plasmid transfected SiHa cell lines were used as controls; lnc-CC3 indicates lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SD of independent experiments, * p

    Techniques Used: Over Expression, Migration, In Vitro, In Situ Hybridization, Microarray, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Cytometry, Colony Assay, Wound Healing Assay, Transwell Assay, Plasmid Preparation, Transfection

    Lnc-CC3 knockdown suppressed migration and invasion in SiHa cells in vitro ( A ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( B ) The shape of lnc-CC3 knockdown SiHa cells changed from elongated to round. Photograph at 100× magnification, scale bar = 200 μm. ( C ) Cell migration capacity was determined by wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification, at the same location in each well. ( D ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Data are expressed as mean ± SD of independent experiments, * p
    Figure Legend Snippet: Lnc-CC3 knockdown suppressed migration and invasion in SiHa cells in vitro ( A ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( B ) The shape of lnc-CC3 knockdown SiHa cells changed from elongated to round. Photograph at 100× magnification, scale bar = 200 μm. ( C ) Cell migration capacity was determined by wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification, at the same location in each well. ( D ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Data are expressed as mean ± SD of independent experiments, * p

    Techniques Used: Migration, In Vitro, Expressing, Quantitative RT-PCR, Wound Healing Assay, Transwell Assay

    Lnc-CC3 promoted EMT in cervical cancer cells by increasing Slug expression ( A ) Lnc-CC3 overexpression increased the expression of Slug and Snail, and lnc-CC3 knockdown increased the expression of E-cadherin and β-catenin. ( B ) Changes in Slug, Snail, E-cadherin, and β-catenin protein levels in SiHa cells. The effects of lnc-CC3 overexpression on Snail and Slug mRNA ( C ) and protein ( D ) levels in CaSki and HeLa cells. The qRT-PCR data were analyzed using the 2 −ΔΔCT method and WB data were quantified by densitometry using ImageJ software. Data are expressed as mean ± SD of independent experiments ( n = 3), * p
    Figure Legend Snippet: Lnc-CC3 promoted EMT in cervical cancer cells by increasing Slug expression ( A ) Lnc-CC3 overexpression increased the expression of Slug and Snail, and lnc-CC3 knockdown increased the expression of E-cadherin and β-catenin. ( B ) Changes in Slug, Snail, E-cadherin, and β-catenin protein levels in SiHa cells. The effects of lnc-CC3 overexpression on Snail and Slug mRNA ( C ) and protein ( D ) levels in CaSki and HeLa cells. The qRT-PCR data were analyzed using the 2 −ΔΔCT method and WB data were quantified by densitometry using ImageJ software. Data are expressed as mean ± SD of independent experiments ( n = 3), * p

    Techniques Used: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Software

    Screening for new genes associated with cervical cancer ( A ) Genbank ID and description of the 7 identified ESTs. ( B ) PCR showed that CC3 was present as a single band in the gel. ( C ) CC3 was expressed in cervical cancer cell lines; expression was higher in SiHa cells than in HeLa and CaSki cells.
    Figure Legend Snippet: Screening for new genes associated with cervical cancer ( A ) Genbank ID and description of the 7 identified ESTs. ( B ) PCR showed that CC3 was present as a single band in the gel. ( C ) CC3 was expressed in cervical cancer cell lines; expression was higher in SiHa cells than in HeLa and CaSki cells.

    Techniques Used: Polymerase Chain Reaction, Expressing

    13) Product Images from "Immunologic Function and Molecular Insight of Recombinant Interleukin-18"

    Article Title: Immunologic Function and Molecular Insight of Recombinant Interleukin-18

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0160321

    Construction of the expression plasmid pPICZα-IL18WT and its mutants. (A) Strategy and schematic presentation of steps involved in the construction of expression plasmid pPICZa-IL18WT. A mature human IL-18 sequence was inserted into the expression plasmid at the Eco RI and Xba I sites. (B) Diagram showing the site-directed mutagenesis method. The mutant-strand was amplified by PCR and a wild-type DNA template was digested by Dpn I. The resulting annealed double-stranded nicked DNA molecules were transformed into E . coli DH5α and the nicked DNA was repaired.
    Figure Legend Snippet: Construction of the expression plasmid pPICZα-IL18WT and its mutants. (A) Strategy and schematic presentation of steps involved in the construction of expression plasmid pPICZa-IL18WT. A mature human IL-18 sequence was inserted into the expression plasmid at the Eco RI and Xba I sites. (B) Diagram showing the site-directed mutagenesis method. The mutant-strand was amplified by PCR and a wild-type DNA template was digested by Dpn I. The resulting annealed double-stranded nicked DNA molecules were transformed into E . coli DH5α and the nicked DNA was repaired.

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Mutagenesis, Amplification, Polymerase Chain Reaction, Transformation Assay

    14) Product Images from "Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification"

    Article Title: Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-206

    Sensitivity of LAMP for TGEV detection . A, LAMP; B, PCR; C, Nest-PCR; 1-7, TGEV RNA sample at 10 -1 , 10 -2 ...10 -7 dilutions respectively; 8. Negative Control.
    Figure Legend Snippet: Sensitivity of LAMP for TGEV detection . A, LAMP; B, PCR; C, Nest-PCR; 1-7, TGEV RNA sample at 10 -1 , 10 -2 ...10 -7 dilutions respectively; 8. Negative Control.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    15) Product Images from "HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species"

    Article Title: HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0232-6

    Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b . Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c . Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined
    Figure Legend Snippet: Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b . Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c . Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined

    Techniques Used: Plasmid Preparation, Construct, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Clone Assay, Sequencing

    16) Product Images from "Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes"

    Article Title: Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067952

    URT pulse-chase assays with model N-end rule substrates in E. coli and V. vulnificus . The set of URT-based 3f DHFR-Ub R48 -X-βgal 3f fusions (X = Val, Leu, Arg, Asp) was assayed for the in vivo degradation of the released (by the yeast Ubp1 DUB) X-βgal proteins in E. coli ( A, B ) and in V. vulnificus ( C, D ) using 35 S-pulse-chases ( A, C ) and their quantification ( B, D ), as described in Materials and Methods. The bands of the 110 kDa X-βgal test proteins and the 33 kDa 3f DHFR-Ub R48 reference protein are indicated on the left. Designations in B and D : squares, Val-βgal; rhombs, Leu-βgal; triangles, Arg-βgal; crosses, Asp-βgal. E . pKP55-X, encoding the S. cerevisiae Ubp1 DUB and 3f DHFR-Ub R48 -X-βgal 3f URT-based fusions. Other notations on the map denote specific bacterial genes. The nucleotide sequences of pKP77 and pKP55-X are available in GenBank (JX181779 and JX181780). In addition, Table S3 contains the nucleotide sequence of pKP55-X.
    Figure Legend Snippet: URT pulse-chase assays with model N-end rule substrates in E. coli and V. vulnificus . The set of URT-based 3f DHFR-Ub R48 -X-βgal 3f fusions (X = Val, Leu, Arg, Asp) was assayed for the in vivo degradation of the released (by the yeast Ubp1 DUB) X-βgal proteins in E. coli ( A, B ) and in V. vulnificus ( C, D ) using 35 S-pulse-chases ( A, C ) and their quantification ( B, D ), as described in Materials and Methods. The bands of the 110 kDa X-βgal test proteins and the 33 kDa 3f DHFR-Ub R48 reference protein are indicated on the left. Designations in B and D : squares, Val-βgal; rhombs, Leu-βgal; triangles, Arg-βgal; crosses, Asp-βgal. E . pKP55-X, encoding the S. cerevisiae Ubp1 DUB and 3f DHFR-Ub R48 -X-βgal 3f URT-based fusions. Other notations on the map denote specific bacterial genes. The nucleotide sequences of pKP77 and pKP55-X are available in GenBank (JX181779 and JX181780). In addition, Table S3 contains the nucleotide sequence of pKP55-X.

    Techniques Used: Pulse Chase, In Vivo, Sequencing

    17) Product Images from "Perturbation Dynamics of the Rumen Microbiota in Response to Exogenous Butyrate"

    Article Title: Perturbation Dynamics of the Rumen Microbiota in Response to Exogenous Butyrate

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029392

    Temporal changes in the relative abundance (% reads) of A) 2 most dominated genera, Prevotella (solid line) and Succininclasticum (dashed line), and B) Ruminobacter and Treponema , in the rumen microbial community of dairy cows in response to butyrate infusion. Error bars represent SD of 4 samples. Normalized percentages represent the number of 16S rRNA gene sequences assigned to a given genus.
    Figure Legend Snippet: Temporal changes in the relative abundance (% reads) of A) 2 most dominated genera, Prevotella (solid line) and Succininclasticum (dashed line), and B) Ruminobacter and Treponema , in the rumen microbial community of dairy cows in response to butyrate infusion. Error bars represent SD of 4 samples. Normalized percentages represent the number of 16S rRNA gene sequences assigned to a given genus.

    Techniques Used:

    18) Product Images from "B-Cell Epitopes in GroEL of Francisella tularensis"

    Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099847

    Ab12, Ab53, and Ab64 bind FtGroEL but only Ab12 and Ab53 crossreact with E. coli . ( A ) ELISA (data from one of two experiments with similar results are shown). Lysates of E. coli BL21 transformed with pET14b vector containing Ft SchuS4 GroEL DNA, Ft LVS GroEL DNA, or no insert (empty vector) were coated on ELISA plates in the left panels, and greater than 10-fold higher concentrations of SchuS4, LVS, or E. coli TG1 lysates were coated on ELISA plates in the right panels. The coated plates were probed with serial dilutions of the indicated mAbs. ( B ) Western blot. Equivalent concentrations of lysates (based on OD 600 of the bacterial cultures) of SchuS4 or of BL21 transformed with SchuS4 rGroEL-vector or empty vector were electrophoresed in preparative 4–15% polyacrylamide gels under denaturing conditions and, after transfer to nitrocellulose, strips were probed with 10 µg/ml of the indicated mAbs. The positions of prestained molecular weight standards (in kDa) are indicated.
    Figure Legend Snippet: Ab12, Ab53, and Ab64 bind FtGroEL but only Ab12 and Ab53 crossreact with E. coli . ( A ) ELISA (data from one of two experiments with similar results are shown). Lysates of E. coli BL21 transformed with pET14b vector containing Ft SchuS4 GroEL DNA, Ft LVS GroEL DNA, or no insert (empty vector) were coated on ELISA plates in the left panels, and greater than 10-fold higher concentrations of SchuS4, LVS, or E. coli TG1 lysates were coated on ELISA plates in the right panels. The coated plates were probed with serial dilutions of the indicated mAbs. ( B ) Western blot. Equivalent concentrations of lysates (based on OD 600 of the bacterial cultures) of SchuS4 or of BL21 transformed with SchuS4 rGroEL-vector or empty vector were electrophoresed in preparative 4–15% polyacrylamide gels under denaturing conditions and, after transfer to nitrocellulose, strips were probed with 10 µg/ml of the indicated mAbs. The positions of prestained molecular weight standards (in kDa) are indicated.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transformation Assay, Plasmid Preparation, Western Blot, Molecular Weight

    19) Product Images from "Ligand-directed profiling of organelles with internalizing phage libraries"

    Article Title: Ligand-directed profiling of organelles with internalizing phage libraries

    Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

    doi: 10.1002/0471140864.ps3004s79

    Cloning strategy to generate the iPhage library. The f88-4 phage vector contains two capsid genes encoding a wild-type (wt) protein VIII (pVIII) and a recombinant protein VIII (rpVIII). The recombinant gene VIII contains a foreign DNA insert with a Hin
    Figure Legend Snippet: Cloning strategy to generate the iPhage library. The f88-4 phage vector contains two capsid genes encoding a wild-type (wt) protein VIII (pVIII) and a recombinant protein VIII (rpVIII). The recombinant gene VIII contains a foreign DNA insert with a Hin

    Techniques Used: Clone Assay, Plasmid Preparation, Recombinant

    20) Product Images from "In vitro nanobody discovery for integral membrane protein targets"

    Article Title: In vitro nanobody discovery for integral membrane protein targets

    Journal: Scientific Reports

    doi: 10.1038/srep06760

    mRNA/cDNA display of Nb-GFP. (a) Nb-GFP-encoding DNA 12 was in vitro transcribed to (b) mRNA, and (c) ligated to a 3′ puromycin (P) containing DNA linker. The shift in molecular weight was tracked on an agarose gel. (d) In vitro translation resulted in displayed mRNA-Nb-GFP covalent fusions (F), detected on a western blot by an increase in molecular weight to ~170 kD, v/s non-displayed Nb-GFP (~15 kD). (e) Fusions (+ve) or untranslated ligations (−ve) were reverse transcribed and (f) binding-selection was performed on Ni-NTA-immobilized purified GFP protein. Binder Nbs were eluted, and sequences were regenerated using PCR. The absence of PCR product from –ve control demonstrates successful antigenic selection. (M – molecular weight markers).
    Figure Legend Snippet: mRNA/cDNA display of Nb-GFP. (a) Nb-GFP-encoding DNA 12 was in vitro transcribed to (b) mRNA, and (c) ligated to a 3′ puromycin (P) containing DNA linker. The shift in molecular weight was tracked on an agarose gel. (d) In vitro translation resulted in displayed mRNA-Nb-GFP covalent fusions (F), detected on a western blot by an increase in molecular weight to ~170 kD, v/s non-displayed Nb-GFP (~15 kD). (e) Fusions (+ve) or untranslated ligations (−ve) were reverse transcribed and (f) binding-selection was performed on Ni-NTA-immobilized purified GFP protein. Binder Nbs were eluted, and sequences were regenerated using PCR. The absence of PCR product from –ve control demonstrates successful antigenic selection. (M – molecular weight markers).

    Techniques Used: In Vitro, Molecular Weight, Agarose Gel Electrophoresis, Western Blot, Binding Assay, Selection, Purification, Polymerase Chain Reaction

    Schematic for mRNA/cDNA display of Nbs. (a) Nb-encoding DNA is in vitro transcribed to (b) mRNA, and (c) ligated to a 3′ puromycin (P) containing DNA linker. (d) Following in vitro translation and fusion formation, (e) reverse transcription is performed to synthesize cDNA. (f) mRNA-cDNA-Nb complexes are exposed to the antigen (Ag) of interest, which is immobolized on a solid-phase affinity matrix. Following washes and elution, PCR is performed to retrieve full-length Nb-encoding amplicons (N/C – N- C-termini of displayed Nb).
    Figure Legend Snippet: Schematic for mRNA/cDNA display of Nbs. (a) Nb-encoding DNA is in vitro transcribed to (b) mRNA, and (c) ligated to a 3′ puromycin (P) containing DNA linker. (d) Following in vitro translation and fusion formation, (e) reverse transcription is performed to synthesize cDNA. (f) mRNA-cDNA-Nb complexes are exposed to the antigen (Ag) of interest, which is immobolized on a solid-phase affinity matrix. Following washes and elution, PCR is performed to retrieve full-length Nb-encoding amplicons (N/C – N- C-termini of displayed Nb).

    Techniques Used: In Vitro, Polymerase Chain Reaction

    Specificity of antigenic selection during mRNA/cDNA display. The mutant library that was constructed based on the Nb-GFP template was displayed using the mRNA/cDNA display method described in this manuscript, and exposed to either GFP or mouse P-glycoprotein (mP-gp), which is an integral membrane transporter protein purified as previously published 18 . Following washes and elution, the binder sequences were regenerated using PCR. The agarose gel shows that for the same input material (library and RT reaction), Nb-specific binders were obtained for selection with GFP as the antigen, 40% sequences of which were confirmed to be Wt Nb-GFP, whereas Nb-specific PCR product was absent following exposure to mP-gp as the antigen, confirming the mRNA/cDNA display method was successful in removing non-specific binder Nbs.
    Figure Legend Snippet: Specificity of antigenic selection during mRNA/cDNA display. The mutant library that was constructed based on the Nb-GFP template was displayed using the mRNA/cDNA display method described in this manuscript, and exposed to either GFP or mouse P-glycoprotein (mP-gp), which is an integral membrane transporter protein purified as previously published 18 . Following washes and elution, the binder sequences were regenerated using PCR. The agarose gel shows that for the same input material (library and RT reaction), Nb-specific binders were obtained for selection with GFP as the antigen, 40% sequences of which were confirmed to be Wt Nb-GFP, whereas Nb-specific PCR product was absent following exposure to mP-gp as the antigen, confirming the mRNA/cDNA display method was successful in removing non-specific binder Nbs.

    Techniques Used: Selection, Mutagenesis, Construct, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    21) Product Images from "Induction of protective immunity against severe acute respiratory syndrome coronavirus (SARS-CoV) infection using highly attenuated recombinant vaccinia virus DIs"

    Article Title: Induction of protective immunity against severe acute respiratory syndrome coronavirus (SARS-CoV) infection using highly attenuated recombinant vaccinia virus DIs

    Journal: Virology

    doi: 10.1016/j.virol.2006.03.020

    Schematic diagram of rDIs constructs expressing SARS-CoV structural proteins. DNA fragments encoding E, M, N and S proteins were inserted into the location of the 15.4 kb deletion in DIs using the vaccinia virus transfer vector pDIsgptmH5. Six rDIs constructs are shown.
    Figure Legend Snippet: Schematic diagram of rDIs constructs expressing SARS-CoV structural proteins. DNA fragments encoding E, M, N and S proteins were inserted into the location of the 15.4 kb deletion in DIs using the vaccinia virus transfer vector pDIsgptmH5. Six rDIs constructs are shown.

    Techniques Used: Construct, Expressing, Plasmid Preparation

    22) Product Images from "Allelic differences of clustered terpene synthases contribute to correlated intra-specific variation of floral and herbivory-induced volatiles in a wild tobacco"

    Article Title: Allelic differences of clustered terpene synthases contribute to correlated intra-specific variation of floral and herbivory-induced volatiles in a wild tobacco

    Journal: bioRxiv

    doi: 10.1101/2020.04.26.062133

    Allelic expression of NaTPS38 in F1 hybrids of Arizona and Utah genotypes. ( a ) Scheme of primers used to measure the allele-specific transcript abundance. Green arrows refer to primers that can specifically amplify the Utah allele and blue arrows refer to primers that can specifically amplify the Arizona allele. Primers (grey arrows) that do not distinguish the two alleles were also used to measure total transcript abundance of NaTPS38 . ( b ) Expression differences of NaTPS38 alleles between F0 and F1 plants. Plants were treated with wounding plus oral secretion (N=5). Mean and SE are shown. Student’s t -test was used to determine differences between F0 and F1 plants. ns refers to no significant difference was found. ( c ) Expression differences of alleles in F1 individuals. Plants were treated with wounding plus oral secretion (N=5). Each dot represents an individual plant. Difference between two alleles was determined by paired Student’s t -test. Blue and green colors indicate alleles from Arizona and Utah genotypes, respectively.
    Figure Legend Snippet: Allelic expression of NaTPS38 in F1 hybrids of Arizona and Utah genotypes. ( a ) Scheme of primers used to measure the allele-specific transcript abundance. Green arrows refer to primers that can specifically amplify the Utah allele and blue arrows refer to primers that can specifically amplify the Arizona allele. Primers (grey arrows) that do not distinguish the two alleles were also used to measure total transcript abundance of NaTPS38 . ( b ) Expression differences of NaTPS38 alleles between F0 and F1 plants. Plants were treated with wounding plus oral secretion (N=5). Mean and SE are shown. Student’s t -test was used to determine differences between F0 and F1 plants. ns refers to no significant difference was found. ( c ) Expression differences of alleles in F1 individuals. Plants were treated with wounding plus oral secretion (N=5). Each dot represents an individual plant. Difference between two alleles was determined by paired Student’s t -test. Blue and green colors indicate alleles from Arizona and Utah genotypes, respectively.

    Techniques Used: Expressing

    Allelic variations of the terpene cluster are associated with differences in floral volatiles and HIPVs among N. attenuata natural accessions. ( a ) Three different haplotypes found from the open reading frame of NaTPS38 from N. attenuata accessions. The amino acid sequence of NaTPS38 from the Utah genotype is set as the reference, and the different haplotype loci are marked in red. The presence of intact NaTPS25 gene in the natural accessions are shown on the right (+, present; -not present). ( b ) The emission of ( E )-α-bergamotene and ( E )-β-ocimene in both flowers and herbivory-induced leaves are different among three NaTPS38 haplotypes. The data for ( E )-α-bergamotene emission were extract from our previous study ( Zhou et al ., 2017 ). Mean and SE are shown. Statistical differences among haplotypes are determined by ANOVA ( P
    Figure Legend Snippet: Allelic variations of the terpene cluster are associated with differences in floral volatiles and HIPVs among N. attenuata natural accessions. ( a ) Three different haplotypes found from the open reading frame of NaTPS38 from N. attenuata accessions. The amino acid sequence of NaTPS38 from the Utah genotype is set as the reference, and the different haplotype loci are marked in red. The presence of intact NaTPS25 gene in the natural accessions are shown on the right (+, present; -not present). ( b ) The emission of ( E )-α-bergamotene and ( E )-β-ocimene in both flowers and herbivory-induced leaves are different among three NaTPS38 haplotypes. The data for ( E )-α-bergamotene emission were extract from our previous study ( Zhou et al ., 2017 ). Mean and SE are shown. Statistical differences among haplotypes are determined by ANOVA ( P

    Techniques Used: Sequencing

    23) Product Images from "DCs facilitate B cell responses against microbial DNA via DC-SIGN"

    Article Title: DCs facilitate B cell responses against microbial DNA via DC-SIGN

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0185580

    CD11c + DCs enhance IFN-α production of CD11c - cells via DC-SIGN. ( A-D ) mRNA expression of monocyte-derived DCs stimulated with EC-DNA ( A ), control ODN, ODN-2216 ( B,D ), or R837 ( C ) after treatment with control, TLR7, TLR9 ( A-C ), or IRF7 siRNA ( D ) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. ( E ) Immunoblot of monocyte-derived DCs or Raji cells whole cell lysate for TLR9. β-actin was used as loading control. ( F ) Analysis of TLR9 expression in monocyte-derived DC culture by flow cytometry. Number adjacent to gates indicates percentage of gated cells. ( G ) mRNA expression of sorted CD11c + DCs and CD11c - cells stimulated with control ODN or ODN-2216 was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with ODN-2216 of CD11c - cells. ( H ) mRNA expression of sorted CD11c + DCs and CD11c - cells stimulated with control ODN or ODN-2216 after treatment with control or DC-SIGN siRNA. Sorted cells were stimulated for 1h, washed and combined for 5h before mRNA expression was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments. Data are collated (mean ± s.d.) of four ( A,B,G ), three ( H ) or two ( C,D ) independent experiments with different donors or are representative of four ( F ) or two ( E ) independent experiments with different donors. *P
    Figure Legend Snippet: CD11c + DCs enhance IFN-α production of CD11c - cells via DC-SIGN. ( A-D ) mRNA expression of monocyte-derived DCs stimulated with EC-DNA ( A ), control ODN, ODN-2216 ( B,D ), or R837 ( C ) after treatment with control, TLR7, TLR9 ( A-C ), or IRF7 siRNA ( D ) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. ( E ) Immunoblot of monocyte-derived DCs or Raji cells whole cell lysate for TLR9. β-actin was used as loading control. ( F ) Analysis of TLR9 expression in monocyte-derived DC culture by flow cytometry. Number adjacent to gates indicates percentage of gated cells. ( G ) mRNA expression of sorted CD11c + DCs and CD11c - cells stimulated with control ODN or ODN-2216 was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with ODN-2216 of CD11c - cells. ( H ) mRNA expression of sorted CD11c + DCs and CD11c - cells stimulated with control ODN or ODN-2216 after treatment with control or DC-SIGN siRNA. Sorted cells were stimulated for 1h, washed and combined for 5h before mRNA expression was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments. Data are collated (mean ± s.d.) of four ( A,B,G ), three ( H ) or two ( C,D ) independent experiments with different donors or are representative of four ( F ) or two ( E ) independent experiments with different donors. *P

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Flow Cytometry

    DC-SIGN binds class A ODN and microbial DNA. ( A , B , D - H ) E . coli DNA ( A , B , D ), human DNA ( D ) or indicated ODNs ( E-H ) were coated on high binding plates and recombinant DC-SIGN binding to coated ligands was measured by ELISA. ( C ) Recombinant DC-SIGN was coated on high binding plates and binding to DNAse-treated or untreated biotin-labeled E . coli DNA or Fucose was measured by ELISA. ( I-K ) Binding of parental Raji cells or Raji cells stably expressing DC-SIGN or Langerin to FITC-labeled E . coli DNA ( I,J ) or FITC-labeled ODN-2216 ( K ) was analyzed by flow cytometry. 10 μg/ml DNA or 5 μM ODN was used in all experiments unless stated otherwise. Data are collated (mean ± s.d.) of four independent experiments ( G ) or representative of at least four ( I ), three ( E ) or two ( A-D,F,H , J , K ) independent experiments (mean ± s.d. of duplicates in A - F,H ). *P
    Figure Legend Snippet: DC-SIGN binds class A ODN and microbial DNA. ( A , B , D - H ) E . coli DNA ( A , B , D ), human DNA ( D ) or indicated ODNs ( E-H ) were coated on high binding plates and recombinant DC-SIGN binding to coated ligands was measured by ELISA. ( C ) Recombinant DC-SIGN was coated on high binding plates and binding to DNAse-treated or untreated biotin-labeled E . coli DNA or Fucose was measured by ELISA. ( I-K ) Binding of parental Raji cells or Raji cells stably expressing DC-SIGN or Langerin to FITC-labeled E . coli DNA ( I,J ) or FITC-labeled ODN-2216 ( K ) was analyzed by flow cytometry. 10 μg/ml DNA or 5 μM ODN was used in all experiments unless stated otherwise. Data are collated (mean ± s.d.) of four independent experiments ( G ) or representative of at least four ( I ), three ( E ) or two ( A-D,F,H , J , K ) independent experiments (mean ± s.d. of duplicates in A - F,H ). *P

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Stable Transfection, Expressing, Flow Cytometry

    DC-SIGN facilitates microbial DNA induced responses. ( A , B,E,F ) mRNA analysis of monocyte-derived DCs stimulated with EC-DNA ( A,E ), ODN-2216 or control ODN ( B,F ) in the presence or absence of IgG1 isotype control or blocking antibodies against DC-SIGN ( A , B ) or after treatment with control or DC-SIGN siRNA ( E,F ) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with IgG1 isotype control or control siRNA. ( C , D ) DC-SIGN expression of monocyte-derived DCs after control or DC-SIGN siRNA treatment was measured by flow cytometry. ( G ) Expression of CD80, CD83 and CD86 expression by monocyte-derived DCs stimulated with control ODN or ODN-2216 in the presence or absence of IgG1 isotype control or blocking antibodies against DC-SIGN. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments. Data are collated (mean ± s.d.) of six ( D ), four ( A,B, ) or three ( E,F ) independent experiments with different donors or are representative of six ( C ) or two ( G ) independent experiments with different donors. *P
    Figure Legend Snippet: DC-SIGN facilitates microbial DNA induced responses. ( A , B,E,F ) mRNA analysis of monocyte-derived DCs stimulated with EC-DNA ( A,E ), ODN-2216 or control ODN ( B,F ) in the presence or absence of IgG1 isotype control or blocking antibodies against DC-SIGN ( A , B ) or after treatment with control or DC-SIGN siRNA ( E,F ) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with IgG1 isotype control or control siRNA. ( C , D ) DC-SIGN expression of monocyte-derived DCs after control or DC-SIGN siRNA treatment was measured by flow cytometry. ( G ) Expression of CD80, CD83 and CD86 expression by monocyte-derived DCs stimulated with control ODN or ODN-2216 in the presence or absence of IgG1 isotype control or blocking antibodies against DC-SIGN. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments. Data are collated (mean ± s.d.) of six ( D ), four ( A,B, ) or three ( E,F ) independent experiments with different donors or are representative of six ( C ) or two ( G ) independent experiments with different donors. *P

    Techniques Used: Derivative Assay, Blocking Assay, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry

    Dendritic cells produce type I IFN or cytokines in response to synthetic and microbial DNA. ( A , B , D , E ) mRNA analysis of monocyte-derived DCs stimulated with EC-DNA ( A , B , D ), human DNA ( D ), ODN-2216 or control ODN ( E ) for indicated time points was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with the highest expression. ( C ) Similar as in ( A ), but EC-DNA was treated with DNAse before stimulation. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments unless stated otherwise. Data are collated (mean ± s.d.) of four ( C ), three ( A , B ) or two ( D , E ) independent experiments with different donors *P
    Figure Legend Snippet: Dendritic cells produce type I IFN or cytokines in response to synthetic and microbial DNA. ( A , B , D , E ) mRNA analysis of monocyte-derived DCs stimulated with EC-DNA ( A , B , D ), human DNA ( D ), ODN-2216 or control ODN ( E ) for indicated time points was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with the highest expression. ( C ) Similar as in ( A ), but EC-DNA was treated with DNAse before stimulation. Cells were stimulated with 10 μg/ml DNA or 5μM ODN in all experiments unless stated otherwise. Data are collated (mean ± s.d.) of four ( C ), three ( A , B ) or two ( D , E ) independent experiments with different donors *P

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing

    Dendritic cells interact with both class A ODN and microbial DNA via DC-SIGN. ( A - D ). Flow cytometry analysis of monocyte-derived DCs incubated with EC-DNA-FITC ( A,B ) or ODN-2216-FITC ( C,D ) for 10 min in the presences or absence of EGTA, IgG1 isotype control or blocking antibodies directed against DC-SIGN. ( E , F ) Confocal imaging of EC-DNA-FITC (green, E ) or ODN-2216-FITC (green, F ), early endosome antigen 1 (EEA1, red), DC-SIGN (turquoise) and DNA (Hoechst, blue) in monocyte-derived DCs stimulated with EC-DNA-FITC ( E ) or ODN-2216-FITC ( F ). 10 μg/ml DNA or 5 μM ODN was used in all experiments. Data are collated (mean ± s.d.) of three ( B , D ) independent experiments with different donors or are representative of at least three ( A,C ) or two ( E,F ) independent experiments with different donors. *P
    Figure Legend Snippet: Dendritic cells interact with both class A ODN and microbial DNA via DC-SIGN. ( A - D ). Flow cytometry analysis of monocyte-derived DCs incubated with EC-DNA-FITC ( A,B ) or ODN-2216-FITC ( C,D ) for 10 min in the presences or absence of EGTA, IgG1 isotype control or blocking antibodies directed against DC-SIGN. ( E , F ) Confocal imaging of EC-DNA-FITC (green, E ) or ODN-2216-FITC (green, F ), early endosome antigen 1 (EEA1, red), DC-SIGN (turquoise) and DNA (Hoechst, blue) in monocyte-derived DCs stimulated with EC-DNA-FITC ( E ) or ODN-2216-FITC ( F ). 10 μg/ml DNA or 5 μM ODN was used in all experiments. Data are collated (mean ± s.d.) of three ( B , D ) independent experiments with different donors or are representative of at least three ( A,C ) or two ( E,F ) independent experiments with different donors. *P

    Techniques Used: Flow Cytometry, Derivative Assay, Incubation, Blocking Assay, Imaging

    24) Product Images from "Acute appendicitis is associated with appendiceal microbiome changes including elevated Campylobacter jejuni levels"

    Article Title: Acute appendicitis is associated with appendiceal microbiome changes including elevated Campylobacter jejuni levels

    Journal: BMJ Open Gastroenterology

    doi: 10.1136/bmjgast-2020-000412

    Using a cut-off of 0.31 pg/µL Campylobacter jejuni DNA levels identified subjects with appendicitis with a specificity of 93.7%, sensitivity of 40.9% and OR of 10.38.
    Figure Legend Snippet: Using a cut-off of 0.31 pg/µL Campylobacter jejuni DNA levels identified subjects with appendicitis with a specificity of 93.7%, sensitivity of 40.9% and OR of 10.38.

    Techniques Used:

    25) Product Images from "Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection"

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    Journal: ACS Omega

    doi: 10.1021/acsomega.0c01181

    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.
    Figure Legend Snippet: Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Techniques Used: DNA Extraction, Lysis, Electrophoresis, Polymerase Chain Reaction, Amplification, Incubation

    Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.
    Figure Legend Snippet: Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Techniques Used: Fluorescence, Microscopy, DNA Extraction, Polymerase Chain Reaction

    DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).
    Figure Legend Snippet: DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Techniques Used: DNA Extraction, Polymerase Chain Reaction, Amplification, DNA Binding Assay, Binding Assay, Purification, Genetically Modified, Transgenic Assay, Modification

    QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.
    Figure Legend Snippet: QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    26) Product Images from "Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase"

    Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    Lane 1: PCR product of Cre gene, lane 2: pET28a-cre plasmid extraction result, lane 3: DNA ladder, lane 4: double digestion of recombinant pET28a-cre by NheI and XhoI. Products were electrophoresed on 0.7% agarose gel.
    Figure Legend Snippet: Lane 1: PCR product of Cre gene, lane 2: pET28a-cre plasmid extraction result, lane 3: DNA ladder, lane 4: double digestion of recombinant pET28a-cre by NheI and XhoI. Products were electrophoresed on 0.7% agarose gel.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Agarose Gel Electrophoresis

    27) Product Images from "Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments"

    Article Title: Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188453

    Sequencing and restriction endonuclease mapping of reactivated scHPXV YFP-gpt::095 clones. a . Pulsed field gel electrophoretic analysis. Three independent HPXV clones plus a VACV (strain WR) control virus were purified and then left either untreated, or digested with Bsa I or Hin dIII. The larger size of HPXV relative to VACV is apparent in the uncut samples, and the absence of nearly all of the Bsa I sites in the HPXV clones is also apparent. The selective manner in which only the Bsa I sites were modified by gene synthesis is illustrated by the retention of all the Hin dIII restriction sites in scHPXV YFP-gpt::095. The Hin dIII digest also demonstrates the similarities between HPXV and VACV as illustrated by the related digestion patterns. The faint DNA bands marked with asterisks (*) are from monkey cell mitochondria, which co-purify with poxvirus particles. b . Sequence reads associated with the hairpin telomeres. The viruses were sequenced using Illumina technology and the genomes assembled using CLC genomics software. A subset of the longest sequencing reads, extending beyond the known end of the HPXV sequence, are shown aligned against a poly·dC template. (This method captures sequences extending beyond the point where the reference sequence ends.) These reads span the entire length of the unfolded hairpins as was provided using synthetic oligonucleotides. Because of the inverted terminal duplications, all of the reads “pile up” together. Both F- and S-forms of the VACV hairpin are detected and the ratio of F- to S-reads in this region was 1.03±0.01 (SEM) in eight different virus-sequencing reactions.
    Figure Legend Snippet: Sequencing and restriction endonuclease mapping of reactivated scHPXV YFP-gpt::095 clones. a . Pulsed field gel electrophoretic analysis. Three independent HPXV clones plus a VACV (strain WR) control virus were purified and then left either untreated, or digested with Bsa I or Hin dIII. The larger size of HPXV relative to VACV is apparent in the uncut samples, and the absence of nearly all of the Bsa I sites in the HPXV clones is also apparent. The selective manner in which only the Bsa I sites were modified by gene synthesis is illustrated by the retention of all the Hin dIII restriction sites in scHPXV YFP-gpt::095. The Hin dIII digest also demonstrates the similarities between HPXV and VACV as illustrated by the related digestion patterns. The faint DNA bands marked with asterisks (*) are from monkey cell mitochondria, which co-purify with poxvirus particles. b . Sequence reads associated with the hairpin telomeres. The viruses were sequenced using Illumina technology and the genomes assembled using CLC genomics software. A subset of the longest sequencing reads, extending beyond the known end of the HPXV sequence, are shown aligned against a poly·dC template. (This method captures sequences extending beyond the point where the reference sequence ends.) These reads span the entire length of the unfolded hairpins as was provided using synthetic oligonucleotides. Because of the inverted terminal duplications, all of the reads “pile up” together. Both F- and S-forms of the VACV hairpin are detected and the ratio of F- to S-reads in this region was 1.03±0.01 (SEM) in eight different virus-sequencing reactions.

    Techniques Used: Sequencing, Clone Assay, Pulsed-Field Gel, Purification, Modification, Software

    Virus design strategy. ( a ) Cloned synthetic DNA fragments used to assemble HPXV. Nine different clones were synthesized spanning all but the first and last 40 bp in GenBank entry DQ792504, each overlapping the adjacent fragment by ~1 kbp. All of the Aar I and Bsa I restriction sites were eliminated from fragments 1A to 7, inclusive, using silent mutations and the same strategy was used to add Ava I and Stu I sites in Frag_2. To facilitate virus recovery a gene encoding a YFP-gpt fusion protein was inserted into Frag_3, at the site of the HPXV thymidine kinase locus. An additional HPXV095 fragment spans the thymidine kinase locus and was subsequently used to delete and replace the YFP-gpt marker using homologous recombination. ( b ) Synthetic hairpin telomeres. Because the HPXV genome was not sequenced to the ends, we substituted two hairpin sequences based upon those reported for VACV strain WR (green coloured nucleotides). These are called “fast” and “slow” forms based upon their electrophoretic properties. The nucleotides coloured in black come from the HPXV genome sequence and provide an element essential for telomere resolution.
    Figure Legend Snippet: Virus design strategy. ( a ) Cloned synthetic DNA fragments used to assemble HPXV. Nine different clones were synthesized spanning all but the first and last 40 bp in GenBank entry DQ792504, each overlapping the adjacent fragment by ~1 kbp. All of the Aar I and Bsa I restriction sites were eliminated from fragments 1A to 7, inclusive, using silent mutations and the same strategy was used to add Ava I and Stu I sites in Frag_2. To facilitate virus recovery a gene encoding a YFP-gpt fusion protein was inserted into Frag_3, at the site of the HPXV thymidine kinase locus. An additional HPXV095 fragment spans the thymidine kinase locus and was subsequently used to delete and replace the YFP-gpt marker using homologous recombination. ( b ) Synthetic hairpin telomeres. Because the HPXV genome was not sequenced to the ends, we substituted two hairpin sequences based upon those reported for VACV strain WR (green coloured nucleotides). These are called “fast” and “slow” forms based upon their electrophoretic properties. The nucleotides coloured in black come from the HPXV genome sequence and provide an element essential for telomere resolution.

    Techniques Used: Clone Assay, Synthesized, Antiviral Assay, Marker, Homologous Recombination, Sequencing

    Characterization of VACV-HPXV hybrid viruses. a . A PCR-based screening approach was used to identify hybrid and reactivated viruses. PCR primers were designed to target both HPXV and VACV ( S2 Table ) and used to amplify DNA segments spanning the Bsa I sites that were disrupted in the synthetic HPXV clones. Following PCR amplification, the products were digested with Bsa I to differentiate VACV sequences (which cut) from HPXV (which do not cut). The VACV/HPXV hybrids exhibit a mix of Bsa I sensitive and resistant sites whereas a reactivated scHPXV YFP-gpt::095 clone is fully Bsa I resistant. b . Sequence mapping of HPXV inserts in VACV strain WR. Virus genomes were sequenced using an Illumina platform, assembled, and LAGAN [ 26 ] and “Base-by-Base” [ 27 ] software were used to align and generate the maps shown. Places where VACV sequences (white) have been replaced by HPXV sequences are colour coded according to the difference. The first hybrid virus (“VACV/HPXV + fragment 3”) was obtained by co-transfecting VACV DNA plus HPXV Frag_3 ( Fig 1 ) into SFV-infected cells. The green-tagged insertion encodes the YFP-gpt selection marker. Clones 1–3 were obtained by purifying the DNA from this first hybrid genome and transfecting it again, along with HPXV fragments 2, 4, 5, and 7, into SFV-infected cells. c . Genomic sequence comparison of scHPXV YFP-gpt::095 to VACV WR.
    Figure Legend Snippet: Characterization of VACV-HPXV hybrid viruses. a . A PCR-based screening approach was used to identify hybrid and reactivated viruses. PCR primers were designed to target both HPXV and VACV ( S2 Table ) and used to amplify DNA segments spanning the Bsa I sites that were disrupted in the synthetic HPXV clones. Following PCR amplification, the products were digested with Bsa I to differentiate VACV sequences (which cut) from HPXV (which do not cut). The VACV/HPXV hybrids exhibit a mix of Bsa I sensitive and resistant sites whereas a reactivated scHPXV YFP-gpt::095 clone is fully Bsa I resistant. b . Sequence mapping of HPXV inserts in VACV strain WR. Virus genomes were sequenced using an Illumina platform, assembled, and LAGAN [ 26 ] and “Base-by-Base” [ 27 ] software were used to align and generate the maps shown. Places where VACV sequences (white) have been replaced by HPXV sequences are colour coded according to the difference. The first hybrid virus (“VACV/HPXV + fragment 3”) was obtained by co-transfecting VACV DNA plus HPXV Frag_3 ( Fig 1 ) into SFV-infected cells. The green-tagged insertion encodes the YFP-gpt selection marker. Clones 1–3 were obtained by purifying the DNA from this first hybrid genome and transfecting it again, along with HPXV fragments 2, 4, 5, and 7, into SFV-infected cells. c . Genomic sequence comparison of scHPXV YFP-gpt::095 to VACV WR.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Amplification, Sequencing, Software, Infection, Selection, Marker

    28) Product Images from "SAM-Dependent Enzyme-Catalysed Pericyclic Reactions in Natural Product Biosynthesis"

    Article Title: SAM-Dependent Enzyme-Catalysed Pericyclic Reactions in Natural Product Biosynthesis

    Journal: Nature

    doi: 10.1038/nature23882

    Enzyme-catalysed pericyclic reactions and the proposed inverse electron demand hetero-Diels-Alder (HDA) reactions in Nature. (a) Examples of enzymatic pericyclic reactions. (b) The structures of natural products containing dihydropyran, which would be biosynthesized by HDA reaction. Variecolortide A is naturally racemic; the relative stereochemistry of epipyridone and leporin B are shown. (c) The putative leporin biosynthetic gene cluster in A. flavus and assignment of encoded genes and biosynthetic pathway of leporins. PKS–NRPS, polyketide synthase–nonribosomal peptide synthetase; TF, transcription factor; MCT, monocarboxylate transporter; SDR, short-chain dehydrogenase/reductase; ER, enoylreductase; OMT, O -methyltransferase. The structures show the relative stereochemistry. (d) Analysis of metabolites from the transformants of A. nidulans . The peak at 12 min correspond to the tetramic acid product that is biosynthesized by LepA (PKS-NRPS) and LepG (ER).
    Figure Legend Snippet: Enzyme-catalysed pericyclic reactions and the proposed inverse electron demand hetero-Diels-Alder (HDA) reactions in Nature. (a) Examples of enzymatic pericyclic reactions. (b) The structures of natural products containing dihydropyran, which would be biosynthesized by HDA reaction. Variecolortide A is naturally racemic; the relative stereochemistry of epipyridone and leporin B are shown. (c) The putative leporin biosynthetic gene cluster in A. flavus and assignment of encoded genes and biosynthetic pathway of leporins. PKS–NRPS, polyketide synthase–nonribosomal peptide synthetase; TF, transcription factor; MCT, monocarboxylate transporter; SDR, short-chain dehydrogenase/reductase; ER, enoylreductase; OMT, O -methyltransferase. The structures show the relative stereochemistry. (d) Analysis of metabolites from the transformants of A. nidulans . The peak at 12 min correspond to the tetramic acid product that is biosynthesized by LepA (PKS-NRPS) and LepG (ER).

    Techniques Used: Helicase-dependent Amplification

    29) Product Images from "Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant"

    Article Title: Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170504

    Bovine, ovine, and caprine CD40 protein sequences have high homology to swine CD40 protein sequence. Alignment of swine, bovine, caprine, and ovine CD40 amino acid sequences. The signal sequence is shown where the consensus sequence is highlighted in green (amino acid 1–19), whereas the consensus sequence of the transmembrane domain is highlighted in red (amino acid 192–215). The percentage identity of the extracellular domains of bovine, ovine, and caprine CD40 protein sequences to that of swine is 74%, 75%, and 75%, respectively.
    Figure Legend Snippet: Bovine, ovine, and caprine CD40 protein sequences have high homology to swine CD40 protein sequence. Alignment of swine, bovine, caprine, and ovine CD40 amino acid sequences. The signal sequence is shown where the consensus sequence is highlighted in green (amino acid 1–19), whereas the consensus sequence of the transmembrane domain is highlighted in red (amino acid 192–215). The percentage identity of the extracellular domains of bovine, ovine, and caprine CD40 protein sequences to that of swine is 74%, 75%, and 75%, respectively.

    Techniques Used: Sequencing

    Reactivity of the mAb 2E4E4 against HEK-293A cells expressing swine or bovine CD40. Evaluation of the mAb 2E4E4 specificity against swine and bovine CD40 was performed by immunocytometric analysis: A. Analysis of mAb 2E4E4 and a defined IgG1 control mAb by PAGE; B. HEK-293A cells transfected with a construct expressing full length swine CD40 and probed with 2E4E4; C. HEK-293A cells transfected with a construct expressing full length bovine CD40 and probed with the mAb 2E4E4; and D. Sham treated HEK-293A cells probed with the mAb 2E4E4. Flow cytometric analysis performed on: E. HEK-293A cells transfected with a construct encoding full length swine CD40; or F. full length bovine CD40 probed with either the mAb 2E4E4 (Red) or IgG1 isotype control (Blue).
    Figure Legend Snippet: Reactivity of the mAb 2E4E4 against HEK-293A cells expressing swine or bovine CD40. Evaluation of the mAb 2E4E4 specificity against swine and bovine CD40 was performed by immunocytometric analysis: A. Analysis of mAb 2E4E4 and a defined IgG1 control mAb by PAGE; B. HEK-293A cells transfected with a construct expressing full length swine CD40 and probed with 2E4E4; C. HEK-293A cells transfected with a construct expressing full length bovine CD40 and probed with the mAb 2E4E4; and D. Sham treated HEK-293A cells probed with the mAb 2E4E4. Flow cytometric analysis performed on: E. HEK-293A cells transfected with a construct encoding full length swine CD40; or F. full length bovine CD40 probed with either the mAb 2E4E4 (Red) or IgG1 isotype control (Blue).

    Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, Transfection, Construct, Flow Cytometry

    Validation of the specificity of the mAb 2E4E4 against CD40 expressed in swine, bovine, ovine, and caprine spleen. Immunohistochemistry performed on: A) swine; B) bovine; C) ovine; and D) caprine spleen tissues probed with the mAb 2E4E4. Background reactivity was tested by probing E) swine, F) bovine, G) ovine, and H) caprine spleen tissues with an IgG1 isotype control mAb.
    Figure Legend Snippet: Validation of the specificity of the mAb 2E4E4 against CD40 expressed in swine, bovine, ovine, and caprine spleen. Immunohistochemistry performed on: A) swine; B) bovine; C) ovine; and D) caprine spleen tissues probed with the mAb 2E4E4. Background reactivity was tested by probing E) swine, F) bovine, G) ovine, and H) caprine spleen tissues with an IgG1 isotype control mAb.

    Techniques Used: Immunohistochemistry

    The mAb 2E4E4 recognized CD40 on stimulated swine and bovine PBMCs. Flow cytometry performed on A) swine and B) bovine PBMCs stimulated (Gold) or not stimulated (Red) with LPS and probed with the mAb 2E4E4. IgG1 isotype control (Blue) was also used to probe LPS-stimulated swine and bovine PBMCs.
    Figure Legend Snippet: The mAb 2E4E4 recognized CD40 on stimulated swine and bovine PBMCs. Flow cytometry performed on A) swine and B) bovine PBMCs stimulated (Gold) or not stimulated (Red) with LPS and probed with the mAb 2E4E4. IgG1 isotype control (Blue) was also used to probe LPS-stimulated swine and bovine PBMCs.

    Techniques Used: Flow Cytometry, Cytometry

    30) Product Images from "Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4"

    Article Title: Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2017.55.2.143

    PCR amplification of T. gondii ROP4 (A) and influenza M1 genes (B). T. gondii ROP4 (1,728 bp) gene was RCR-amplified from cDNA synthesized using a Prime Script 1st Strain cDNA Synthesis Kit using total RNA extracted from T. gondii RH. Influenza M1 gene was PCR amplified from total RNA extract from influenza virus (A/PR/8/34). M, DNA marker; TgROP4, T. gondii ROP4; M1, influenza M1.
    Figure Legend Snippet: PCR amplification of T. gondii ROP4 (A) and influenza M1 genes (B). T. gondii ROP4 (1,728 bp) gene was RCR-amplified from cDNA synthesized using a Prime Script 1st Strain cDNA Synthesis Kit using total RNA extracted from T. gondii RH. Influenza M1 gene was PCR amplified from total RNA extract from influenza virus (A/PR/8/34). M, DNA marker; TgROP4, T. gondii ROP4; M1, influenza M1.

    Techniques Used: Polymerase Chain Reaction, Amplification, Synthesized, Marker

    31) Product Images from "The Argi system: one-step purification of proteins tagged with arginine-rich cell-penetrating peptides"

    Article Title: The Argi system: one-step purification of proteins tagged with arginine-rich cell-penetrating peptides

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02432-6

    Analysis of arginine-rich peptide-tagged protein purification from E. coli total protein extract using AR aptamer-based chromatography. The recombinant proteins: GFP-R 6 ( A ), R 6 -GST ( B ), R 6 -PCNA ( C ), GFP-R 8 ( D ), R 8 -GST ( E ), R 8 -PCNA ( F ), GFP-Tat 49–57 ( G ), Tat 49–57 -GST ( H ) and Tat 49–57 -PCNA ( I ) were overexpressed and purified from E. coli total protein extract using AR aptamer. Lanes: M) molecular weight marker; 1) non-induced BL21-CodonPlus(DE3)-RIL[pET29a arginine-rich peptide-tagged protein] cells; 2) induced BL21-CodonPlus(DE3)-RIL [pET29a arginine-rich peptide-tagged protein] cells; 3) cell lysate; 4) 4 μg of protein sample eluted from AR aptamer-based resin using ARGI buffer supplemented with GuHCl. The samples were denatured and separated on 12% SDS-PAGE followed by Coomassie brilliant blue staining. This is one of three independent experiments which is representative.
    Figure Legend Snippet: Analysis of arginine-rich peptide-tagged protein purification from E. coli total protein extract using AR aptamer-based chromatography. The recombinant proteins: GFP-R 6 ( A ), R 6 -GST ( B ), R 6 -PCNA ( C ), GFP-R 8 ( D ), R 8 -GST ( E ), R 8 -PCNA ( F ), GFP-Tat 49–57 ( G ), Tat 49–57 -GST ( H ) and Tat 49–57 -PCNA ( I ) were overexpressed and purified from E. coli total protein extract using AR aptamer. Lanes: M) molecular weight marker; 1) non-induced BL21-CodonPlus(DE3)-RIL[pET29a arginine-rich peptide-tagged protein] cells; 2) induced BL21-CodonPlus(DE3)-RIL [pET29a arginine-rich peptide-tagged protein] cells; 3) cell lysate; 4) 4 μg of protein sample eluted from AR aptamer-based resin using ARGI buffer supplemented with GuHCl. The samples were denatured and separated on 12% SDS-PAGE followed by Coomassie brilliant blue staining. This is one of three independent experiments which is representative.

    Techniques Used: Protein Purification, Chromatography, Recombinant, Purification, Molecular Weight, Marker, SDS Page, Staining

    32) Product Images from "Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid"

    Article Title: Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid

    Journal: Nature Communications

    doi: 10.1038/ncomms15828

    Production of 2,4-dihydroxybutyrate by engineered E. coli strains. Cells were cultivated in shake flasks on mineral medium containing 20 g per l glucose. Values correspond to concentrations after 24 h of cultivation. Error bars represent STDV from at least two replicate experiments. All strains were derived from E. coli K-12 substr. MG1655. All plasmids were derived from the pACT3 medium-copy number plasmid. pHOM expresses the genes encoding the homoserine pathway enzymes AK: Ec-LysC E250K, ASD: Ec-Asd, HSD: Sc-Hom6. pDHB expresses the genes encoding the DHB pathway enzymes MK: Ec-LysC V115A:E119S:E250K:E434V, MSD: Bs-Asd E218Q, MSR: Ms-Ssr H39R:N43H. pDHB-ppc* additionally expresses the malate-insensitive PEP carboxylase mutant Ppc K620S . pDHBopt-ppc* has optimized ribosome binding sites in front of each DHB pathway gene. pDHBopt-ppc*(Ec-asd*) and pDHBopt-ppc*(Mj-asd*) express, respectively, the Ec-Asd E241Q or Mj-Asd E210Q mutant enzymes instead of Bs-Asd E218Q .
    Figure Legend Snippet: Production of 2,4-dihydroxybutyrate by engineered E. coli strains. Cells were cultivated in shake flasks on mineral medium containing 20 g per l glucose. Values correspond to concentrations after 24 h of cultivation. Error bars represent STDV from at least two replicate experiments. All strains were derived from E. coli K-12 substr. MG1655. All plasmids were derived from the pACT3 medium-copy number plasmid. pHOM expresses the genes encoding the homoserine pathway enzymes AK: Ec-LysC E250K, ASD: Ec-Asd, HSD: Sc-Hom6. pDHB expresses the genes encoding the DHB pathway enzymes MK: Ec-LysC V115A:E119S:E250K:E434V, MSD: Bs-Asd E218Q, MSR: Ms-Ssr H39R:N43H. pDHB-ppc* additionally expresses the malate-insensitive PEP carboxylase mutant Ppc K620S . pDHBopt-ppc* has optimized ribosome binding sites in front of each DHB pathway gene. pDHBopt-ppc*(Ec-asd*) and pDHBopt-ppc*(Mj-asd*) express, respectively, the Ec-Asd E241Q or Mj-Asd E210Q mutant enzymes instead of Bs-Asd E218Q .

    Techniques Used: Derivative Assay, Plasmid Preparation, Mass Spectrometry, Mutagenesis, Binding Assay

    33) Product Images from "Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli"

    Article Title: Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli

    Journal: International Journal of Genomics

    doi: 10.1155/2018/9235605

    The calibrator can be applied to the induction system xylS- Pm . (a) xylS promoters ( PxylS ), xylS protein, and Pm promoter were cloned into the plasmid pMR1, which contains a short-lived GFP variant. (b) xylS- Pm calibration in LB solid medium with 1000 μ M of 3MBz added. This calibration was performed in BW25113 wt strains. (c) Pjx promoter activity analyzed by 8 hours of experiment by monitoring GFPlva expression using pMR1 constructions. (d) GFP expression profile for 7 different 3MBz concentrations for Pjx and xylS- Pm in pMR1 vector, 4.5 hours after the induction. Solid lines represent the average values calculated using data from three independent experiments for wild type, Δihf , and Δfis strains, while dashed lines represent the upper and lower limits of standard deviations.
    Figure Legend Snippet: The calibrator can be applied to the induction system xylS- Pm . (a) xylS promoters ( PxylS ), xylS protein, and Pm promoter were cloned into the plasmid pMR1, which contains a short-lived GFP variant. (b) xylS- Pm calibration in LB solid medium with 1000 μ M of 3MBz added. This calibration was performed in BW25113 wt strains. (c) Pjx promoter activity analyzed by 8 hours of experiment by monitoring GFPlva expression using pMR1 constructions. (d) GFP expression profile for 7 different 3MBz concentrations for Pjx and xylS- Pm in pMR1 vector, 4.5 hours after the induction. Solid lines represent the average values calculated using data from three independent experiments for wild type, Δihf , and Δfis strains, while dashed lines represent the upper and lower limits of standard deviations.

    Techniques Used: Clone Assay, Plasmid Preparation, Variant Assay, Activity Assay, Expressing

    34) Product Images from "Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK"

    Article Title: Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.7b01497

    Reaction of NosI with ATP and MIA. ATP (1 mM) and MIA (1 mM) were incubated with 2 (red triangles), 4 (black triangles), 8 μM (green triangles) NosI in the absence of NosK for varying lengths of time (15 to 800 s), and MIA-AMP was measured by LC-MS as a relative integrated response (A). Amplitudes from each of the time courses plotted as a function of NosI concentration show a linear correlation (B). The reactions were conducted in an ice–water bath.
    Figure Legend Snippet: Reaction of NosI with ATP and MIA. ATP (1 mM) and MIA (1 mM) were incubated with 2 (red triangles), 4 (black triangles), 8 μM (green triangles) NosI in the absence of NosK for varying lengths of time (15 to 800 s), and MIA-AMP was measured by LC-MS as a relative integrated response (A). Amplitudes from each of the time courses plotted as a function of NosI concentration show a linear correlation (B). The reactions were conducted in an ice–water bath.

    Techniques Used: Incubation, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

    HPLC analysis of holo NosJ upon reaction with ATP, MIA, and NosI. (A) apo NosJ; (B) holo NosJ; (C) holo NosJ + ATP and MIA; (D) holo NosJ + NosI and MIA; (E) holo NosJ + NosI and ATP; and (F) holo NosJ + NosI, MIA, and ATP (complete reaction).
    Figure Legend Snippet: HPLC analysis of holo NosJ upon reaction with ATP, MIA, and NosI. (A) apo NosJ; (B) holo NosJ; (C) holo NosJ + ATP and MIA; (D) holo NosJ + NosI and MIA; (E) holo NosJ + NosI and ATP; and (F) holo NosJ + NosI, MIA, and ATP (complete reaction).

    Techniques Used: High Performance Liquid Chromatography

    Characterization of NosJ and NosK. Panel 1 shows as-purified NosJ (theoretical mass is 8717.63 Da). Panel 2 displays the mass shift corresponding to the transfer of 4′-phosphopantetheine to NosJ to produce holo NosJ (theoretical mass is 9057.96 Da). Panel 3 shows the NosI-dependent transfer of MIA to NosJ to produce MIA-holo NosJ (theoretical mass is 9215.13 Da). Finally, upon incubation with NosK, MIA-holo NosJ is reverted back to holo NosJ due to MIA transfer to NosK (Panel 4). The proposed reaction scheme is shown at bottom left.
    Figure Legend Snippet: Characterization of NosJ and NosK. Panel 1 shows as-purified NosJ (theoretical mass is 8717.63 Da). Panel 2 displays the mass shift corresponding to the transfer of 4′-phosphopantetheine to NosJ to produce holo NosJ (theoretical mass is 9057.96 Da). Panel 3 shows the NosI-dependent transfer of MIA to NosJ to produce MIA-holo NosJ (theoretical mass is 9215.13 Da). Finally, upon incubation with NosK, MIA-holo NosJ is reverted back to holo NosJ due to MIA transfer to NosK (Panel 4). The proposed reaction scheme is shown at bottom left.

    Techniques Used: Purification, Incubation

    HPLC analysis of the acylation of NosK. NosK, blue; NosK + NosJ, black; NosK + NosJ + NosI, pink; NosK Ser102Ala variant, red; NosK Ser102Ala variant + NosJ + NosI, green.
    Figure Legend Snippet: HPLC analysis of the acylation of NosK. NosK, blue; NosK + NosJ, black; NosK + NosJ + NosI, pink; NosK Ser102Ala variant, red; NosK Ser102Ala variant + NosJ + NosI, green.

    Techniques Used: High Performance Liquid Chromatography, Variant Assay

    35) Product Images from "Distribution of Florfenicol Resistance Genes fexA and cfr among Chloramphenicol-Resistant Staphylococcus Isolates"

    Article Title: Distribution of Florfenicol Resistance Genes fexA and cfr among Chloramphenicol-Resistant Staphylococcus Isolates

    Journal:

    doi: 10.1128/AAC.50.4.1156-1163.2006

    (a) PCR amplicons specific for the detection of transposase genes tnpA (lane 1), tnpB (lane 2), and tnpC (lane 3), the linkage between tnpB-fexA (lane 4), an internal 5,741-bp fragment representing the almost complete Tn 558 (lane 5), and a circular intermediate
    Figure Legend Snippet: (a) PCR amplicons specific for the detection of transposase genes tnpA (lane 1), tnpB (lane 2), and tnpC (lane 3), the linkage between tnpB-fexA (lane 4), an internal 5,741-bp fragment representing the almost complete Tn 558 (lane 5), and a circular intermediate

    Techniques Used: Polymerase Chain Reaction

    36) Product Images from "SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of ?F, ?E, and ?G but Does Not Control Solvent Production in Clostridium acetobutylicum ATCC 824 ▿ ATCC 824 ▿ ‡"

    Article Title: SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of ?F, ?E, and ?G but Does Not Control Solvent Production in Clostridium acetobutylicum ATCC 824 ▿ ATCC 824 ▿ ‡

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.05474-11

    Southern blot confirmation of pKOSPOIIE integration site. (A) NdeI-digested genomic DNA from C. acetobutylicum WT and SPOIIEKO hybridized with a probe for the 2nd homologous region. Lane 1, NdeI-digested SPOIIEKO DNA; lane 2, BamHI-digested pKOSPOIIE;
    Figure Legend Snippet: Southern blot confirmation of pKOSPOIIE integration site. (A) NdeI-digested genomic DNA from C. acetobutylicum WT and SPOIIEKO hybridized with a probe for the 2nd homologous region. Lane 1, NdeI-digested SPOIIEKO DNA; lane 2, BamHI-digested pKOSPOIIE;

    Techniques Used: Southern Blot

    37) Product Images from "Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells †"

    Article Title: Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells †

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.1.535-545.2003

    Metabolic labeling of polypeptides synthesized in virus-infected SF9 and SL2 cells. Cells (10 5 ) were infected at an MOI of 20. At 45 h postinfection, metabolic labeling was performed with a 35 S-labeled methionine-cysteine mixture. Analysis was done with extracts from cells infected with Ac M NPV (lanes 2 and 7), vAcSL2 (lanes 3 and 8), vBgl3 (lanes 4 and 9), and vHsp-1 (lanes 5 and 10) and with mock-infected SF9 cells (lane 1) and SL2 cells (lane 6). Ph, location of Ac M NPV polyhedrin.
    Figure Legend Snippet: Metabolic labeling of polypeptides synthesized in virus-infected SF9 and SL2 cells. Cells (10 5 ) were infected at an MOI of 20. At 45 h postinfection, metabolic labeling was performed with a 35 S-labeled methionine-cysteine mixture. Analysis was done with extracts from cells infected with Ac M NPV (lanes 2 and 7), vAcSL2 (lanes 3 and 8), vBgl3 (lanes 4 and 9), and vHsp-1 (lanes 5 and 10) and with mock-infected SF9 cells (lane 1) and SL2 cells (lane 6). Ph, location of Ac M NPV polyhedrin.

    Techniques Used: Labeling, Synthesized, Infection

    38) Product Images from "Inactivation of ?F in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes ?E and ?G Protein Expression but Does Not Block Solvent Formation ▿ Protein Expression but Does Not Block Solvent Formation ▿ †"

    Article Title: Inactivation of ?F in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes ?E and ?G Protein Expression but Does Not Block Solvent Formation ▿ Protein Expression but Does Not Block Solvent Formation ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00088-11

    (A) Southern blot of HindIII-digested genomic DNA from C. acetobutylicum WT and FKO1 strains. Lane 1, WT DNA; lane 2, pKOSIGF; lane 3, FKO1 DNA; lane L, 2-log ladder from NEB. The membrane was exposed for 10 min. (B) Diagrams of the bands expected from the WT and FKO1 strains and of the probed region. The four short arrows represent CAC2302, CAC2303, CAC2304, and CAC2305 and are not drawn to scale.
    Figure Legend Snippet: (A) Southern blot of HindIII-digested genomic DNA from C. acetobutylicum WT and FKO1 strains. Lane 1, WT DNA; lane 2, pKOSIGF; lane 3, FKO1 DNA; lane L, 2-log ladder from NEB. The membrane was exposed for 10 min. (B) Diagrams of the bands expected from the WT and FKO1 strains and of the probed region. The four short arrows represent CAC2302, CAC2303, CAC2304, and CAC2305 and are not drawn to scale.

    Techniques Used: Southern Blot

    Integration of pKOSIGF into the C. acetobutylicum genome to disrupt the sigF gene. (A) The pKOSIGF vector with two regions of homology for sigF and the orientation of sigF in the genome. (B) A single-crossover event with pKOSIGF via the first region of homology resulted in the integration of the entire plasmid. The P ptb promoter and rho-independent terminators (RIT) are indicated. The presumable P spoIIA promoter is also indicated. (C) Primers used to confirm the integration and orientation of pKOSIGF. (D) PCR results for the WT and FKO1 strains demonstrating the integration of pKOSIGF through the first region of homology.
    Figure Legend Snippet: Integration of pKOSIGF into the C. acetobutylicum genome to disrupt the sigF gene. (A) The pKOSIGF vector with two regions of homology for sigF and the orientation of sigF in the genome. (B) A single-crossover event with pKOSIGF via the first region of homology resulted in the integration of the entire plasmid. The P ptb promoter and rho-independent terminators (RIT) are indicated. The presumable P spoIIA promoter is also indicated. (C) Primers used to confirm the integration and orientation of pKOSIGF. (D) PCR results for the WT and FKO1 strains demonstrating the integration of pKOSIGF through the first region of homology.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction

    39) Product Images from "Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase"

    Article Title: Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004714

    Purification of recombinant Tsf- Tb DHFR-TS from E . coli . A. SDS-PAGE gel stained with Coomassie blue. Lane 1. Clarified thyA - E . coli lysate expressing Tsf- Tb DHFR-TS. Lane 2. Glycerol-diluted lysate treated with TEV protease. Lane 3. Methotrexate agarose elution. Proteins indicated by arrows identified by mass spectrometry to be Tsf- Tb DHFR-TS, cleaved Tb DHFR-TS, and the Tsf tag. B. Size exclusion chromatography of purified product. Closed circles: DHFR activity. Dashed line: absorbance at 280 nm.
    Figure Legend Snippet: Purification of recombinant Tsf- Tb DHFR-TS from E . coli . A. SDS-PAGE gel stained with Coomassie blue. Lane 1. Clarified thyA - E . coli lysate expressing Tsf- Tb DHFR-TS. Lane 2. Glycerol-diluted lysate treated with TEV protease. Lane 3. Methotrexate agarose elution. Proteins indicated by arrows identified by mass spectrometry to be Tsf- Tb DHFR-TS, cleaved Tb DHFR-TS, and the Tsf tag. B. Size exclusion chromatography of purified product. Closed circles: DHFR activity. Dashed line: absorbance at 280 nm.

    Techniques Used: Purification, Recombinant, SDS Page, Staining, Expressing, Mass Spectrometry, Size-exclusion Chromatography, Activity Assay

    40) Product Images from "Niche-Partitioning of Prochlorococcus Populations in a Stratified Water Column in the Eastern North Atlantic Ocean †"

    Article Title: Niche-Partitioning of Prochlorococcus Populations in a Stratified Water Column in the Eastern North Atlantic Ocean †

    Journal: Applied and Environmental Microbiology

    doi:

    Representative dot blots showing the specificity of hybridization of each genotype-specific oligonucleotide (HLI, HLII, LL, MIT9303, and SS120) to arrays of control DNA samples ( E. coli , Prochlorococcus sp. strains MED4, NATL1, TATL2, MIT9303, and SS120, Prochlorococcus 16S rDNA clone ENATL4, and Synechococcus sp. strain WH8103). EUB, eubacterial probe.
    Figure Legend Snippet: Representative dot blots showing the specificity of hybridization of each genotype-specific oligonucleotide (HLI, HLII, LL, MIT9303, and SS120) to arrays of control DNA samples ( E. coli , Prochlorococcus sp. strains MED4, NATL1, TATL2, MIT9303, and SS120, Prochlorococcus 16S rDNA clone ENATL4, and Synechococcus sp. strain WH8103). EUB, eubacterial probe.

    Techniques Used: Hybridization

    Vertical distribution of the HLI- and LL-adapted Prochlorococcus 16S rDNA genotypes in two different water columns in the eastern North Atlantic, revealed by dot blot hybridization. In the upper panels are graphs showing the differential hybridization of the HLI and LL probes to oxygenic phototroph 16S rDNA sequences PCR amplified from different depths in depth profiles 1 (A) and 2 (B), together with the temperature and fluorescence data. Dot blots corresponding to the graphs from which the relative hybridization of each genotype-specific probe was quantified are shown in the lower panels, together with the PAR measured at each depth. EUB, eubacterial probe.
    Figure Legend Snippet: Vertical distribution of the HLI- and LL-adapted Prochlorococcus 16S rDNA genotypes in two different water columns in the eastern North Atlantic, revealed by dot blot hybridization. In the upper panels are graphs showing the differential hybridization of the HLI and LL probes to oxygenic phototroph 16S rDNA sequences PCR amplified from different depths in depth profiles 1 (A) and 2 (B), together with the temperature and fluorescence data. Dot blots corresponding to the graphs from which the relative hybridization of each genotype-specific probe was quantified are shown in the lower panels, together with the PAR measured at each depth. EUB, eubacterial probe.

    Techniques Used: Dot Blot, Hybridization, Polymerase Chain Reaction, Amplification, Fluorescence

    DGGE analysis of the distribution of Prochlorococcus 16S rDNA genotypes in depth profiles 1 (A) and 2 (B). 16S rDNA sequences amplified from cultured Prochlorococcus strains were used as controls: panel A, lane 1, MED4; lane 2, ENATL4; lane 3, SS120; lane 4, TATL2; lane 5, mixture of MED4, SS120, ENATL4, and TATL2; lane 6, 10 m; lane 7, 20 m; lane 8, 30 m; lane 9, 40 m; lane 10, 50 m; lane 11, 60 m; and lane 12, 70 m; panel B, lane 1, MED4; lane 2, ENATL4; lane 3, SS120; lane 4, NATL1; lane 5, TATL1; lane 6, TATL2; lane 7, 10 m; lane 8, 30 m; lane 9, 40 m; lane 10, 50 m; lane 11, 60 m; lane 12, 70 m; lane 13, 90 m; and lane 14, 110 m.
    Figure Legend Snippet: DGGE analysis of the distribution of Prochlorococcus 16S rDNA genotypes in depth profiles 1 (A) and 2 (B). 16S rDNA sequences amplified from cultured Prochlorococcus strains were used as controls: panel A, lane 1, MED4; lane 2, ENATL4; lane 3, SS120; lane 4, TATL2; lane 5, mixture of MED4, SS120, ENATL4, and TATL2; lane 6, 10 m; lane 7, 20 m; lane 8, 30 m; lane 9, 40 m; lane 10, 50 m; lane 11, 60 m; and lane 12, 70 m; panel B, lane 1, MED4; lane 2, ENATL4; lane 3, SS120; lane 4, NATL1; lane 5, TATL1; lane 6, TATL2; lane 7, 10 m; lane 8, 30 m; lane 9, 40 m; lane 10, 50 m; lane 11, 60 m; lane 12, 70 m; lane 13, 90 m; and lane 14, 110 m.

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Amplification, Cell Culture

    Related Articles

    Clone Assay:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The mRNA template was amplified with Qβ replicase and subsequently reverse transcribed and PCR-amplified (Superscript III™ RT-PCR; Invitrogen) prior to blunt-end cloning into pPCR-Script Amp SK (+) (Stratagene), transformed into E. coli strain HB2151, and clones chosen at random were sequenced. .. The control reaction was processed as outlined above, however, the mRNA was not amplified with Qβ replicase.

    Article Title: Exploring potassium-dependent GTP hydrolysis in TEES family GTPases
    Article Snippet: .. 2.2 Cloning, expression and purification of proteins DNA sequences corresponding to wild type YphC, GD1-YphC, GD2-YphC and EngB were PCR amplified from the Bacillus subtilis genome ( ) using pfu DNA polymerase (Fermentas). ..

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

    Amplification:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The mRNA template was amplified with Qβ replicase and subsequently reverse transcribed and PCR-amplified (Superscript III™ RT-PCR; Invitrogen) prior to blunt-end cloning into pPCR-Script Amp SK (+) (Stratagene), transformed into E. coli strain HB2151, and clones chosen at random were sequenced. .. The control reaction was processed as outlined above, however, the mRNA was not amplified with Qβ replicase.

    Article Title: Exploring potassium-dependent GTP hydrolysis in TEES family GTPases
    Article Snippet: .. 2.2 Cloning, expression and purification of proteins DNA sequences corresponding to wild type YphC, GD1-YphC, GD2-YphC and EngB were PCR amplified from the Bacillus subtilis genome ( ) using pfu DNA polymerase (Fermentas). ..

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells
    Article Snippet: .. DNA was amplified using the linker as a primer to generate a sufficient amount to clone into the pCR II-TOPO vector using TOPO TA Cloning Kit with One Shot Max Efficiency DH5α-T1 E. coli according to the manufacturer's suggested protocol (Invitrogen). .. Competent E. coli cells were transformed by heat shock and plated on agarose plates containing ampicillin and S-gal (Sigma).

    TA Cloning:

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells
    Article Snippet: .. DNA was amplified using the linker as a primer to generate a sufficient amount to clone into the pCR II-TOPO vector using TOPO TA Cloning Kit with One Shot Max Efficiency DH5α-T1 E. coli according to the manufacturer's suggested protocol (Invitrogen). .. Competent E. coli cells were transformed by heat shock and plated on agarose plates containing ampicillin and S-gal (Sigma).

    Construct:

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

    Purification:

    Article Title: Exploring potassium-dependent GTP hydrolysis in TEES family GTPases
    Article Snippet: .. 2.2 Cloning, expression and purification of proteins DNA sequences corresponding to wild type YphC, GD1-YphC, GD2-YphC and EngB were PCR amplified from the Bacillus subtilis genome ( ) using pfu DNA polymerase (Fermentas). ..

    Polymerase Chain Reaction:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The mRNA template was amplified with Qβ replicase and subsequently reverse transcribed and PCR-amplified (Superscript III™ RT-PCR; Invitrogen) prior to blunt-end cloning into pPCR-Script Amp SK (+) (Stratagene), transformed into E. coli strain HB2151, and clones chosen at random were sequenced. .. The control reaction was processed as outlined above, however, the mRNA was not amplified with Qβ replicase.

    Article Title: Exploring potassium-dependent GTP hydrolysis in TEES family GTPases
    Article Snippet: .. 2.2 Cloning, expression and purification of proteins DNA sequences corresponding to wild type YphC, GD1-YphC, GD2-YphC and EngB were PCR amplified from the Bacillus subtilis genome ( ) using pfu DNA polymerase (Fermentas). ..

    Article Title: Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes
    Article Snippet: .. The PCR products were ligated into the promoterless pBlue-TOPO and pPROBE-NT vectors and transformed into E. coli strain, Top10 (Invitrogen Technologies, Carlsbad, CA) and DH5α strain, respectively [ ]. .. One clone each in forward and reverse orientations was selected for the genes 14 and 19 in the pBlue-TOPO plasmid.

    Article Title: ARID1A Alterations Are Associated with FGFR3-Wild Type, Poor-Prognosis, Urothelial Bladder Tumors
    Article Snippet: .. Briefly, exons 2–20 were separately PCR-amplified with AccuPrime Taq DNA polymerase High Fidelity (Invitrogen) on DNA from bladder cancer cell lines (RT112, VMCUB-3, MGH-U3, UM-UC-3 and UM-UC-17) and fresh tumor tissue sections containing > 60% neoplastic cells. .. PCR amplimers from each sample were equimolarly pooled and fragmented to a range of 100–300 bp (Covaris S2 shearing instrument).

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells
    Article Snippet: .. DNA was amplified using the linker as a primer to generate a sufficient amount to clone into the pCR II-TOPO vector using TOPO TA Cloning Kit with One Shot Max Efficiency DH5α-T1 E. coli according to the manufacturer's suggested protocol (Invitrogen). .. Competent E. coli cells were transformed by heat shock and plated on agarose plates containing ampicillin and S-gal (Sigma).

    Expressing:

    Article Title: Exploring potassium-dependent GTP hydrolysis in TEES family GTPases
    Article Snippet: .. 2.2 Cloning, expression and purification of proteins DNA sequences corresponding to wild type YphC, GD1-YphC, GD2-YphC and EngB were PCR amplified from the Bacillus subtilis genome ( ) using pfu DNA polymerase (Fermentas). ..

    Article Title: Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes
    Article Snippet: .. Subsequent to restriction endonuclease digestion with Asc 1 and Pme 1, the product was ligated into the lentiviral expression vector, pLenti6.3-MCS/V5 DEST (Invitrogen), which was used to transform E. coli DH5α cells. .. Positive recombinant colonies (named pLenti6.3-EGFP-EGFR-miR and pLenti6.3-EGFP-bFGFR-miR) were screened and identified by PCR, Asc 1 and Pme 1 digestion and DNA sequencing.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The mRNA template was amplified with Qβ replicase and subsequently reverse transcribed and PCR-amplified (Superscript III™ RT-PCR; Invitrogen) prior to blunt-end cloning into pPCR-Script Amp SK (+) (Stratagene), transformed into E. coli strain HB2151, and clones chosen at random were sequenced. .. The control reaction was processed as outlined above, however, the mRNA was not amplified with Qβ replicase.

    Transformation Assay:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The mRNA template was amplified with Qβ replicase and subsequently reverse transcribed and PCR-amplified (Superscript III™ RT-PCR; Invitrogen) prior to blunt-end cloning into pPCR-Script Amp SK (+) (Stratagene), transformed into E. coli strain HB2151, and clones chosen at random were sequenced. .. The control reaction was processed as outlined above, however, the mRNA was not amplified with Qβ replicase.

    Article Title: Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes
    Article Snippet: .. The PCR products were ligated into the promoterless pBlue-TOPO and pPROBE-NT vectors and transformed into E. coli strain, Top10 (Invitrogen Technologies, Carlsbad, CA) and DH5α strain, respectively [ ]. .. One clone each in forward and reverse orientations was selected for the genes 14 and 19 in the pBlue-TOPO plasmid.

    Plasmid Preparation:

    Article Title: Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes
    Article Snippet: .. Subsequent to restriction endonuclease digestion with Asc 1 and Pme 1, the product was ligated into the lentiviral expression vector, pLenti6.3-MCS/V5 DEST (Invitrogen), which was used to transform E. coli DH5α cells. .. Positive recombinant colonies (named pLenti6.3-EGFP-EGFR-miR and pLenti6.3-EGFP-bFGFR-miR) were screened and identified by PCR, Asc 1 and Pme 1 digestion and DNA sequencing.

    Article Title: SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis
    Article Snippet: .. Plasmid construction The protein encoding region of SMA1 was PCR-amplified with primers SMA1CDS-F and SMA1CDS-R, cloned into PCR8/GW/TOPO cloning vector (Invitrogen) and subsequently subcloned into pMDC43 binary vector to generate 35S::GFP-SMA1 construct. .. To generate the bimolecular fluorescence complementation (BiFC) construct for SMA1, SMA1 cDNA was inserted into the multiple clone sites of pSAT4-cCFP-C vector.

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells
    Article Snippet: .. DNA was amplified using the linker as a primer to generate a sufficient amount to clone into the pCR II-TOPO vector using TOPO TA Cloning Kit with One Shot Max Efficiency DH5α-T1 E. coli according to the manufacturer's suggested protocol (Invitrogen). .. Competent E. coli cells were transformed by heat shock and plated on agarose plates containing ampicillin and S-gal (Sigma).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher rt pcr rna
    Fbxl13 and Drc7 are evolutionarily conserved and testis-enriched genes. (A) Model of DRC localization, based on Chlamydomonas [ 13 , 14 ]. The numbers shown in the N-DRC correspond to each DRC component. FBXL13 (DRC6) and DRC7 are located next to TCTE1 (DRC5). (B) The expression of mouse Fbxl13 and Drc7 was examined by <t>RT-PCR</t> using <t>RNA</t> isolated from various organs. Both Fbxl13 and Drc7 are testis-enriched with weak expression detected in other tissues. Actb was used as a loading control. (C) The expression of Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from testis at various postnatal days. Actb was used as a loading control.
    Rt Pcr Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 525 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rt pcr rna/product/Thermo Fisher
    Average 99 stars, based on 525 article reviews
    Price from $9.99 to $1999.99
    rt pcr rna - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher e coli bl21
    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli <t>BL21</t> lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21/product/Thermo Fisher
    Average 94 stars, based on 151 article reviews
    Price from $9.99 to $1999.99
    e coli bl21 - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    89
    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/top10 escherichia coli/product/Thermo Fisher
    Average 89 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    top10 escherichia coli - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    89
    Thermo Fisher e coli bw25113
    MqsA decreases EPS production. (a) Colony morphology of strains grown on salt-free CR plates containing 1 mM IPTG for 7 days. Red color indicated curli/cellulose production and scale bars represent 1 cm. Empty vector: <t>BW25113</t> Δ mqsRA /pBS(Kan); MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA ; and Δ csgD : BW25113 Δ csgD . (b) The amount of Congo red bound to planktonic cells at various time points. Error bars denote standard deviation ( n = 2).
    E Coli Bw25113, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bw25113/product/Thermo Fisher
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli bw25113 - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    Image Search Results


    Fbxl13 and Drc7 are evolutionarily conserved and testis-enriched genes. (A) Model of DRC localization, based on Chlamydomonas [ 13 , 14 ]. The numbers shown in the N-DRC correspond to each DRC component. FBXL13 (DRC6) and DRC7 are located next to TCTE1 (DRC5). (B) The expression of mouse Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from various organs. Both Fbxl13 and Drc7 are testis-enriched with weak expression detected in other tissues. Actb was used as a loading control. (C) The expression of Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from testis at various postnatal days. Actb was used as a loading control.

    Journal: PLoS Genetics

    Article Title: Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice

    doi: 10.1371/journal.pgen.1008585

    Figure Lengend Snippet: Fbxl13 and Drc7 are evolutionarily conserved and testis-enriched genes. (A) Model of DRC localization, based on Chlamydomonas [ 13 , 14 ]. The numbers shown in the N-DRC correspond to each DRC component. FBXL13 (DRC6) and DRC7 are located next to TCTE1 (DRC5). (B) The expression of mouse Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from various organs. Both Fbxl13 and Drc7 are testis-enriched with weak expression detected in other tissues. Actb was used as a loading control. (C) The expression of Fbxl13 and Drc7 was examined by RT-PCR using RNA isolated from testis at various postnatal days. Actb was used as a loading control.

    Article Snippet: Isolation of RNA and RT-PCR RNA was isolated and purified from multiple adult tissues of C57BL/6N mice at different ages with TRIzol (15596018, Thermo Fisher Scientific).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: DNA Extraction, Lysis, Electrophoresis, Polymerase Chain Reaction, Amplification, Incubation

    Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: Fluorescence, Microscopy, DNA Extraction, Polymerase Chain Reaction

    DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: DNA Extraction, Polymerase Chain Reaction, Amplification, DNA Binding Assay, Binding Assay, Purification, Genetically Modified, Transgenic Assay, Modification

    QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: Real-time Polymerase Chain Reaction, Amplification

    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    MqsA decreases EPS production. (a) Colony morphology of strains grown on salt-free CR plates containing 1 mM IPTG for 7 days. Red color indicated curli/cellulose production and scale bars represent 1 cm. Empty vector: BW25113 Δ mqsRA /pBS(Kan); MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA ; and Δ csgD : BW25113 Δ csgD . (b) The amount of Congo red bound to planktonic cells at various time points. Error bars denote standard deviation ( n = 2).

    Journal: Scientific Reports

    Article Title: Antitoxin MqsA Represses Curli Formation Through the Master Biofilm Regulator CsgD

    doi: 10.1038/srep03186

    Figure Lengend Snippet: MqsA decreases EPS production. (a) Colony morphology of strains grown on salt-free CR plates containing 1 mM IPTG for 7 days. Red color indicated curli/cellulose production and scale bars represent 1 cm. Empty vector: BW25113 Δ mqsRA /pBS(Kan); MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA ; and Δ csgD : BW25113 Δ csgD . (b) The amount of Congo red bound to planktonic cells at various time points. Error bars denote standard deviation ( n = 2).

    Article Snippet: Electrophoretic mobility shift assay (EMSA) Gene promoters were PCR-amplified from the genomic DNA of E. coli BW25113 and 3′-labeled with biotin (Biotin 3′-End DNA Labeling kit, Thermo Scientific, Waltham, MA).

    Techniques: Plasmid Preparation, Standard Deviation

    Curli and cellulose are reduced in MqsA-producing cells. Curli production was assayed from cells in 2-day old colonies on agar plates with 1 mM IPTG, and imaged using SEM. Empty vector: BW25113 Δ mqsRA /pBS(Kan) and MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA . For each strain, 3 independent colonies were examined, and an image from one representative colony is shown. Scale bars represent 2 μm (left) and 3 μm (right).

    Journal: Scientific Reports

    Article Title: Antitoxin MqsA Represses Curli Formation Through the Master Biofilm Regulator CsgD

    doi: 10.1038/srep03186

    Figure Lengend Snippet: Curli and cellulose are reduced in MqsA-producing cells. Curli production was assayed from cells in 2-day old colonies on agar plates with 1 mM IPTG, and imaged using SEM. Empty vector: BW25113 Δ mqsRA /pBS(Kan) and MqsA: BW25113 Δ mqsRA /pBS(Kan)- mqsA . For each strain, 3 independent colonies were examined, and an image from one representative colony is shown. Scale bars represent 2 μm (left) and 3 μm (right).

    Article Snippet: Electrophoretic mobility shift assay (EMSA) Gene promoters were PCR-amplified from the genomic DNA of E. coli BW25113 and 3′-labeled with biotin (Biotin 3′-End DNA Labeling kit, Thermo Scientific, Waltham, MA).

    Techniques: Plasmid Preparation