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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
Double Stranded Plasmid Dna Molecules Pbr322 Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
Double Stranded Plasmid Dna Molecules, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
Plasmid Dna Molecule Pnl1.1.Cmv, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
Plasmid φx174 Rf Dna Molecule, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
Naked Plasmid Dna Molecule, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic <t>DNA.</t> (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.
Plasmids Dna Molecules Fosmids Description Source, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic DNA. (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.

Journal: ACS Synthetic Biology

Article Title: Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

doi: 10.1021/acssynbio.3c00355

Figure Lengend Snippet: Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic DNA. (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.

Article Snippet: The following plasmids were procured from Addgene and used as template DNA molecules to create the plasmid constructs used in this work: 1066 pBabe puroL PTEN was a gift from William Sellers (Addgene plasmid # 10785; http://n2t.net/addgene:10785 ; RRID:Addgene_10785); pCDNA3_UnaG-Flag-Sec61B was a gift from Hyun-Woo Rhee (Addgene plasmid # 83413; http://n2t.net/addgene:83413 ; RRID:Addgene_83413); pLEX-MCS-ASC-GFP was a gift from Christian Stehlik (Addgene plasmid # 73957; http://n2t.net/addgene:73957 ; RRID:Addgene_73957); pcDNA3.1(+)_SpyCatcher-6aa-sfCherry2(1–10) was a gift from Bo Huang (Addgene plasmid # 117484; http://n2t.net/addgene:117484 ; RRID:Addgene_117484); gRNA_Cloning Vector was a gift from George Church (Addgene plasmid # 41824; http://n2t.net/addgene:41824 ; RRID:Addgene_41824); pSFFV_sfCherry3C(1–10) was a gift from Bo Huang (Addgene plasmid # 117482; http://n2t.net/addgene:117482 ; RRID:Addgene_117482); pGP-CMV-jGCaMP7c variant 1513 was a gift from Douglas Kim & GENIE Project (Addgene plasmid # 105320; http://n2t.net/addgene:105320 ; RRID:Addgene_105320); and pLNCX2-STIM1 was a gift from Shengyu Yang (Addgene plasmid # 89817; http://n2t.net/addgene:89817 ; RRID:Addgene_89817).

Techniques: Sequencing, Plasmid Preparation, Generated, Construct, Control, Flow Cytometry, Fluorescence, Transfection, Illumina Sequencing, Amplification, Binding Assay, DNA Extraction, Transgenic Assay, Derivative Assay, Virus