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g2 plasmidsaurus whole plasmid nanopore sequencing  (Plasmidsaurus)


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    Plasmidsaurus g2 plasmidsaurus whole plasmid nanopore sequencing
    (A) MS2 genome maps of the constructs tested in this series, showing <t>sequence</t> differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, <t>G2</t> denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.
    G2 Plasmidsaurus Whole Plasmid Nanopore Sequencing, supplied by Plasmidsaurus, 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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    Average 99 stars, based on 1 article reviews
    g2 plasmidsaurus whole plasmid nanopore sequencing - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "A reference genome without a virus: cDNA reconstruction reveals the provenance and function of the MS2 phage sequence"

    Article Title: A reference genome without a virus: cDNA reconstruction reveals the provenance and function of the MS2 phage sequence

    Journal: bioRxiv

    doi: 10.64898/2026.09.18.752701

    (A) MS2 genome maps of the constructs tested in this series, showing sequence differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.
    Figure Legend Snippet: (A) MS2 genome maps of the constructs tested in this series, showing sequence differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.

    Techniques Used: Construct, Sequencing, Control, Transformation Assay, Expressing, Cloning, Plasmid Preparation, Selection, Standard Deviation

    (A) Schematic of the assay used to evaluate MS2 cDNA constructs. Plasmids were transformed into F- E. coli host strains, and outgrowth cultures were sampled at two time points. At 1 hr post-outgrowth, aliquots of live transformed cells were removed to quantify colony-forming units (CFU) as a control for plasmid uptake and transformation efficiency. At 3 hr post-outgrowth, culture supernatants were collected and assayed for plaque-forming units (PFU) on an F+ host to quantify phage production. G1 indicates plasmid tested here after recovery following a single transformation from the original sequence-verified stock (G0). G2 indicates plasmid recovered after an additional propagation cycle from the archived strain carrying G1. (B) Phage production from cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. The T7 expression system used NEB T7 Express cells, which provide T7 RNA polymerase. Induction was not required. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. (D) Performed identically to sets in panel B experiment but with G2 plasmids. (E) Performed identically to sets in panel C experiment but with G2 plasmids. (F) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the G1 constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. Data represent 2-3 independent biological replicates, and error bars indicate standard deviation. ND= not detected.
    Figure Legend Snippet: (A) Schematic of the assay used to evaluate MS2 cDNA constructs. Plasmids were transformed into F- E. coli host strains, and outgrowth cultures were sampled at two time points. At 1 hr post-outgrowth, aliquots of live transformed cells were removed to quantify colony-forming units (CFU) as a control for plasmid uptake and transformation efficiency. At 3 hr post-outgrowth, culture supernatants were collected and assayed for plaque-forming units (PFU) on an F+ host to quantify phage production. G1 indicates plasmid tested here after recovery following a single transformation from the original sequence-verified stock (G0). G2 indicates plasmid recovered after an additional propagation cycle from the archived strain carrying G1. (B) Phage production from cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. The T7 expression system used NEB T7 Express cells, which provide T7 RNA polymerase. Induction was not required. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. (D) Performed identically to sets in panel B experiment but with G2 plasmids. (E) Performed identically to sets in panel C experiment but with G2 plasmids. (F) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the G1 constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. Data represent 2-3 independent biological replicates, and error bars indicate standard deviation. ND= not detected.

    Techniques Used: Construct, Transformation Assay, Control, Plasmid Preparation, Sequencing, Expressing, Cloning, Selection, Standard Deviation

    Related Articles

    Sequencing:

    Article Title: Whole-genome sequencing identifies a c.1282C > T missense variant in Taurine Transporter (TauT) associated with taurine-mediated dilated cardiomyopathy in a family of domestic shorthair cats.
    Article Snippet: Whole-genome sequencing identifies a c.1282C > T missense variant in Taurine Transporter (TauT) associated with taurinemediated dilated cardiomyopathy in a family of domestic shorthair cats Victor N. Rivas, Lisa M. Freeman, Esha M. Srinivasan, Andi Morgan, Melissa Daeschner, John E. Rush, Ronald H.L.. Li, Sandra M. Losa, Josue Gonzalez, Connor J. Thonen-Fleck, Kailyn E. Brown, E. Javier Lopez Soto, Joshua A. Stern PII: S0022-2828(26)00147-1 DOI: https://doi.org/10.1016/j.yjmcc.2026.09.001 Reference: YJMCC 10076 To appear in: Journal of Molecular and Cellular Cardiology Received date: 24 March 2026 Revised date: 3 September 2026 Accepted date: 8 September 2026 Please cite this article as: V.N.. Rivas, L.M.

    Article Title: Growth capacity and mortality burden shape microbial persistence under osmotic stress in Bacteroides thetaiotaomicron
    Article Snippet: .. Plasmids were purified using a QIAprep Spin Miniprep Kit and sequence verified via whole-plasmid sequencing (Plasmidsaurus). ..

    Article Title: Longitudinal dynamics and lineage cross-reactivity of the human antibody response to West Nile virus.
    Article Snippet: .. Following sequence confirmation by whole-plasmid sequencing (Plasmidsaurus), the construct was transformed into BL21(DE3) cells (New England Biolabs). ..

    Article Title: Synonymous HTT CAA/CCA-loss variants associated with early Huntington disease onset enhance toxicity beyond somatic instability
    Article Snippet: Briefly, assembly fragments were designed with 20-40 bp binding overlaps and generated with a combination of polymerase chain reaction (PCR) HotStarTaq DNA Polymerase (Qiagen, Cat#203203) and synthetic fragments generated by Twist Biosciences (San Francisco, CA, USA). .. Plasmids were transformed into MAX Efficiency Stbl2 Competent Cells (Invitrogen, Cat#10268019), isolated using QIAprep Spin Miniprep Kit (Qiagen, Cat#27104), and screened using whole plasmid sequencing through Nanopore sequencing (Plasmidsaurus, Oregon, USA). ..

    Article Title: Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination
    Article Snippet: Point mutations and truncations were generated with Q5 PCR (NEB, M0491L), Gibson Assembly (NEB, E5510S), or site-directed mutagenesis kits Q5 (NEB, E0554S) or QuikChange Lightning (Agilent, 210519) on plasmids carrying the gene of interest. .. All constructs were verified by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY). .. pRIT2-ssb1-mutant plasmids were generated and sequence verified by Genscript (Piscataway, NJ).

    Article Title: Fission yeast RPA-TERT-Tpz1TPP1 complex promotes telomere extension and suppresses telomere recombination.
    Article Snippet: Point mutations and truncations were generated with Q5 PCR (NEB, M0491L), Gibson Assembly (NEB, E5510S), or site-directed mutagenesis kits Q5 (NEB, E0554S) or QuikChange Lightning (Agilent, 210519) on plasmids carrying the gene of interest. .. All constructs were verified by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY). .. pRIT2ssb1-mutant plasmids were generated and sequence verified by Genscript (Piscataway, NJ).

    Article Title: Omics-guided engineering of CHO cells reveals host targets to support AAV production
    Article Snippet: .. Successful clones were confirmed by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY, USA), and high-purity plasmid DNA was prepared using the NucleoBond Xtra Midi kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. .. CHO-S cells from Life Technologies were cultivated and expanded in disposable polycarbonate 125 mL shake flasks equipped with vented caps (Corning Life Sciences, Tewksbury, MA, USA) in CD CHO media supplemented with 8 mM GlutaMAX (Gibco, Life Technologies, Thermo Fisher Scientific, USA) and 0.1% Pluronic F-68 Non-ionic Surfactant (Gibco, Life Technologies).

    other:

    Article Title: Focused ultrasound-mediated lipid nanoparticle delivery for brain gene editing
    Article Snippet: Oligonucleotides were synthesized by Integrated DNA Technologies (IDT).

    Purification:

    Article Title: Growth capacity and mortality burden shape microbial persistence under osmotic stress in Bacteroides thetaiotaomicron
    Article Snippet: .. Plasmids were purified using a QIAprep Spin Miniprep Kit and sequence verified via whole-plasmid sequencing (Plasmidsaurus). ..

    Construct:

    Article Title: Longitudinal dynamics and lineage cross-reactivity of the human antibody response to West Nile virus.
    Article Snippet: .. Following sequence confirmation by whole-plasmid sequencing (Plasmidsaurus), the construct was transformed into BL21(DE3) cells (New England Biolabs). ..

    Article Title: Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination
    Article Snippet: Point mutations and truncations were generated with Q5 PCR (NEB, M0491L), Gibson Assembly (NEB, E5510S), or site-directed mutagenesis kits Q5 (NEB, E0554S) or QuikChange Lightning (Agilent, 210519) on plasmids carrying the gene of interest. .. All constructs were verified by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY). .. pRIT2-ssb1-mutant plasmids were generated and sequence verified by Genscript (Piscataway, NJ).

    Article Title: Fission yeast RPA-TERT-Tpz1TPP1 complex promotes telomere extension and suppresses telomere recombination.
    Article Snippet: Point mutations and truncations were generated with Q5 PCR (NEB, M0491L), Gibson Assembly (NEB, E5510S), or site-directed mutagenesis kits Q5 (NEB, E0554S) or QuikChange Lightning (Agilent, 210519) on plasmids carrying the gene of interest. .. All constructs were verified by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY). .. pRIT2ssb1-mutant plasmids were generated and sequence verified by Genscript (Piscataway, NJ).

    Transformation Assay:

    Article Title: Longitudinal dynamics and lineage cross-reactivity of the human antibody response to West Nile virus.
    Article Snippet: .. Following sequence confirmation by whole-plasmid sequencing (Plasmidsaurus), the construct was transformed into BL21(DE3) cells (New England Biolabs). ..

    Article Title: Synonymous HTT CAA/CCA-loss variants associated with early Huntington disease onset enhance toxicity beyond somatic instability
    Article Snippet: Briefly, assembly fragments were designed with 20-40 bp binding overlaps and generated with a combination of polymerase chain reaction (PCR) HotStarTaq DNA Polymerase (Qiagen, Cat#203203) and synthetic fragments generated by Twist Biosciences (San Francisco, CA, USA). .. Plasmids were transformed into MAX Efficiency Stbl2 Competent Cells (Invitrogen, Cat#10268019), isolated using QIAprep Spin Miniprep Kit (Qiagen, Cat#27104), and screened using whole plasmid sequencing through Nanopore sequencing (Plasmidsaurus, Oregon, USA). ..

    Isolation:

    Article Title: Synonymous HTT CAA/CCA-loss variants associated with early Huntington disease onset enhance toxicity beyond somatic instability
    Article Snippet: Briefly, assembly fragments were designed with 20-40 bp binding overlaps and generated with a combination of polymerase chain reaction (PCR) HotStarTaq DNA Polymerase (Qiagen, Cat#203203) and synthetic fragments generated by Twist Biosciences (San Francisco, CA, USA). .. Plasmids were transformed into MAX Efficiency Stbl2 Competent Cells (Invitrogen, Cat#10268019), isolated using QIAprep Spin Miniprep Kit (Qiagen, Cat#27104), and screened using whole plasmid sequencing through Nanopore sequencing (Plasmidsaurus, Oregon, USA). ..

    Plasmid Preparation:

    Article Title: Synonymous HTT CAA/CCA-loss variants associated with early Huntington disease onset enhance toxicity beyond somatic instability
    Article Snippet: Briefly, assembly fragments were designed with 20-40 bp binding overlaps and generated with a combination of polymerase chain reaction (PCR) HotStarTaq DNA Polymerase (Qiagen, Cat#203203) and synthetic fragments generated by Twist Biosciences (San Francisco, CA, USA). .. Plasmids were transformed into MAX Efficiency Stbl2 Competent Cells (Invitrogen, Cat#10268019), isolated using QIAprep Spin Miniprep Kit (Qiagen, Cat#27104), and screened using whole plasmid sequencing through Nanopore sequencing (Plasmidsaurus, Oregon, USA). ..

    Article Title: Omics-guided engineering of CHO cells reveals host targets to support AAV production
    Article Snippet: .. Successful clones were confirmed by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY, USA), and high-purity plasmid DNA was prepared using the NucleoBond Xtra Midi kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. .. CHO-S cells from Life Technologies were cultivated and expanded in disposable polycarbonate 125 mL shake flasks equipped with vented caps (Corning Life Sciences, Tewksbury, MA, USA) in CD CHO media supplemented with 8 mM GlutaMAX (Gibco, Life Technologies, Thermo Fisher Scientific, USA) and 0.1% Pluronic F-68 Non-ionic Surfactant (Gibco, Life Technologies).

    Nanopore Sequencing:

    Article Title: Synonymous HTT CAA/CCA-loss variants associated with early Huntington disease onset enhance toxicity beyond somatic instability
    Article Snippet: Briefly, assembly fragments were designed with 20-40 bp binding overlaps and generated with a combination of polymerase chain reaction (PCR) HotStarTaq DNA Polymerase (Qiagen, Cat#203203) and synthetic fragments generated by Twist Biosciences (San Francisco, CA, USA). .. Plasmids were transformed into MAX Efficiency Stbl2 Competent Cells (Invitrogen, Cat#10268019), isolated using QIAprep Spin Miniprep Kit (Qiagen, Cat#27104), and screened using whole plasmid sequencing through Nanopore sequencing (Plasmidsaurus, Oregon, USA). ..

    Clone Assay:

    Article Title: Omics-guided engineering of CHO cells reveals host targets to support AAV production
    Article Snippet: .. Successful clones were confirmed by whole-plasmid sequencing (Plasmidsaurus, Louisville, KY, USA), and high-purity plasmid DNA was prepared using the NucleoBond Xtra Midi kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. .. CHO-S cells from Life Technologies were cultivated and expanded in disposable polycarbonate 125 mL shake flasks equipped with vented caps (Corning Life Sciences, Tewksbury, MA, USA) in CD CHO media supplemented with 8 mM GlutaMAX (Gibco, Life Technologies, Thermo Fisher Scientific, USA) and 0.1% Pluronic F-68 Non-ionic Surfactant (Gibco, Life Technologies).



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    (A) MS2 genome maps of the constructs tested in this series, showing <t>sequence</t> differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, <t>G2</t> denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.
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    (A) MS2 genome maps of the constructs tested in this series, showing <t>sequence</t> differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, <t>G2</t> denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.
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    (A) MS2 genome maps of the constructs tested in this series, showing <t>sequence</t> differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, <t>G2</t> denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.
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    (A) MS2 genome maps of the constructs tested in this series, showing <t>sequence</t> differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, <t>G2</t> denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.
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    Image Search Results


    (A) MS2 genome maps of the constructs tested in this series, showing sequence differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.

    Journal: bioRxiv

    Article Title: A reference genome without a virus: cDNA reconstruction reveals the provenance and function of the MS2 phage sequence

    doi: 10.64898/2026.09.18.752701

    Figure Lengend Snippet: (A) MS2 genome maps of the constructs tested in this series, showing sequence differences relative to the current MS2 reference genome (NC_001417.2; total number of differences indicated for each construct). All constructs in this figure contained the same 5′ leak-control architecture: a lac operator ( lacO ), a hammerhead ribozyme (HHR), and an upstream rrnB T1 terminator positioned 51 bp before the T7 promoter. Synonymous mutations are shown as nucleotide changes in gray font, whereas non-synonymous changes are shown as amino-acid changes in black font. Unless otherwise indicated, plasmids tested in panels B–D were G1 plasmids, defined as plasmids tested after recovery from a single transformation of the original sequence-verified stock (G0). (B) Phage production from the indicated cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. Where indicated, G2 denotes plasmids recovered after an additional propagation cycle from the archived strain carrying G1. (D) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. All constructs shown in panels B–D were assayed using the same workflow described in . Points represent independent biological replicates. For G1 constructs in panels B and C, each biological replicate was measured with two technical replicates; for G2 constructs included in panels B and C, four biological replicates were measured. Error bars indicate standard deviation. For the G1–G2 comparisons, both conventional parametric and nonparametric analyses gave the same qualitative result: no significant difference was detected un er the conditions tested. ND , not detected.

    Article Snippet: Both pJLC295- and pJLC298-derived plasmids could also still be successfully sequenced at G2 (Plasmidsaurus whole-plasmid nanopore sequencing).

    Techniques: Construct, Sequencing, Control, Transformation Assay, Expressing, Cloning, Plasmid Preparation, Selection, Standard Deviation

    (A) Schematic of the assay used to evaluate MS2 cDNA constructs. Plasmids were transformed into F- E. coli host strains, and outgrowth cultures were sampled at two time points. At 1 hr post-outgrowth, aliquots of live transformed cells were removed to quantify colony-forming units (CFU) as a control for plasmid uptake and transformation efficiency. At 3 hr post-outgrowth, culture supernatants were collected and assayed for plaque-forming units (PFU) on an F+ host to quantify phage production. G1 indicates plasmid tested here after recovery following a single transformation from the original sequence-verified stock (G0). G2 indicates plasmid recovered after an additional propagation cycle from the archived strain carrying G1. (B) Phage production from cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. The T7 expression system used NEB T7 Express cells, which provide T7 RNA polymerase. Induction was not required. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. (D) Performed identically to sets in panel B experiment but with G2 plasmids. (E) Performed identically to sets in panel C experiment but with G2 plasmids. (F) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the G1 constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. Data represent 2-3 independent biological replicates, and error bars indicate standard deviation. ND= not detected.

    Journal: bioRxiv

    Article Title: A reference genome without a virus: cDNA reconstruction reveals the provenance and function of the MS2 phage sequence

    doi: 10.64898/2026.09.18.752701

    Figure Lengend Snippet: (A) Schematic of the assay used to evaluate MS2 cDNA constructs. Plasmids were transformed into F- E. coli host strains, and outgrowth cultures were sampled at two time points. At 1 hr post-outgrowth, aliquots of live transformed cells were removed to quantify colony-forming units (CFU) as a control for plasmid uptake and transformation efficiency. At 3 hr post-outgrowth, culture supernatants were collected and assayed for plaque-forming units (PFU) on an F+ host to quantify phage production. G1 indicates plasmid tested here after recovery following a single transformation from the original sequence-verified stock (G0). G2 indicates plasmid recovered after an additional propagation cycle from the archived strain carrying G1. (B) Phage production from cDNA constructs in the T7 expression system at 3 hr post-outgrowth, measured as plaque-forming units (PFU) in culture supernatants. The T7 expression system used NEB T7 Express cells, which provide T7 RNA polymerase. Induction was not required. (C) Transformation control for the same constructs at 1 hr post-outgrowth, measured as colony-forming units (CFU) from live transformed cells in the T7 expression system. (D) Performed identically to sets in panel B experiment but with G2 plasmids. (E) Performed identically to sets in panel C experiment but with G2 plasmids. (F) Free phage detected in supernatants of the non-T7 cloning strain NEB 5-alpha after overnight growth under plasmid selection, reflecting basal leakiness of the G1 constructs during plasmid maintenance in the absence of a T7 RNA polymerase expression background. Data represent 2-3 independent biological replicates, and error bars indicate standard deviation. ND= not detected.

    Article Snippet: Both pJLC295- and pJLC298-derived plasmids could also still be successfully sequenced at G2 (Plasmidsaurus whole-plasmid nanopore sequencing).

    Techniques: Construct, Transformation Assay, Control, Plasmid Preparation, Sequencing, Expressing, Cloning, Selection, Standard Deviation