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

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