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

Journal: bioRxiv

Article Title: Firewalled synthetic commensal blocks horizontal gene transfer in the gut

doi: 10.64898/2026.09.17.752456

Figure Lengend Snippet: ( a ) The creation of Syn-Ec F 1 through the stepwise reassignment of serine TCA and TCG codons. First, we deleted RF1 and replaced Syn61’s tRNA Ser(UGA) encoded by serT with an engineered bacteriophage-derived v tRNA Leu(CGA) . Next, we performed laboratory evolution to adapt cells to their modified genetic code and select an optimal reassignment for TCA codons. Only mutable tRNAs that establish an amino-acid-swapped genetic code are displayed. Finally, we deleted serU (tRNA Ser(CGA) ), eliminating residual serine decoding of TCG codons and creating Syn-Ec F 1. ( b ) Syn-Ec F 1 translates TCG codons as leucine and TCA codons as alanine, and ( c ) confers resistance to an array of laboratory and environmental bacteriophages, including REP1−12, that infect Syn61Δ3 3 . Phage infection assays were performed in n =3 independent replicates. Limit of detection = 10 PFU/ml. Bars represent mean bacteriophage titer after 24 hr growth on the corresponding strain, Syn61Δ3 or Syn-Ec F 1; error bars represent standard deviation. ( d – e ) The modified genetic code of Syn-Ec F 1 decodes TCG codons as leucine, while TCA codons are translated as alanine. The amino acid identity of the translated TCG codon ( d ) and TCA codon ( e ) within elastin (16TCG/16TCA) –sfGFP–His 6 was confirmed by LC-MS/MS from Syn-Ec F 1 cells. The figure shows the amino acid sequence and LC-MS/MS spectrum of the analyzed elastin (16TCG/16TCA) peptide. Green star indicates a missed trypsin cleavage in the LC-MS/MS-detected peptide.

Article Snippet: Sequencing of the chromosomal tRNA Ala(UGA) and tRNA Ala(CGA) regions was performed by first PCR amplifying the target region followed by amplicon Nanopore sequencing (Plasmidsaurus, USA).

Techniques: Derivative Assay, Modification, Infection, Standard Deviation, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Firewalled synthetic commensal blocks horizontal gene transfer in the gut

doi: 10.64898/2026.09.17.752456

Figure Lengend Snippet: ( a ) Animals ( n =10/group) independently pre-colonized with Syn-Ec F 1 and E. coli Nissle 1917 were gavaged with an 8-plex mixture of laboratory and environmental bacteriophages, followed by daily fecal sampling for 5 days and then an additional time point at day 18 for bacterial CFU and viral PFU titer evaluation. Plots represent geometric mean; shaded area indicates 95% CI. n.d. represents not detected (limit of detection = 10 PFU/fecal pellet). ( b ) Laboratory co-evolution between bacteriophages and viral-and E. coli cellular-tRNA-based amino-acid-swapped genetic code-bearing cells. Liquid cultures of Syn-Ec F 1 and its bacterial-tRNA Ala(CGA) -based variant were infected with an 8-plex mixture of laboratory and environmental bacteriophages, followed by transfers and phage titer analysis every 3 days. Phage co-evolution experiments were performed in three independent biological replicates; limit of detection = 10 PFU/ml ( i.e. , 500 PFU/flask). ( c ) Sequencing read coverage of the REP12 phage mutant (REP12 Mut ) detected in the cellular-tRNA-based firewall + phage co-evolution experiment with a partially triplicated tRNA operon containing the viral tRNA Ser(UGA) . tRNAs are marked with magenta; viral tRNA Ser(UGA) is highlighted. ( d ) Bacteriophage titer of the REP12 environmental phage isolate and its evolved derivative (REP12 Mut ) following growth on Syn61Δ3 and Syn-Ec F 1. n.d. indicates not detected; limit of detection = 10 PFU/ml. ( e ) Sequencing the swapped-code-establishing E. coli tRNA Ala(UGA) from the co-evolved E. coli cells revealed a G39→A anticodon stem-loop mutation within tRNA Ala(UGA) . tRNA structure was predicted using AlphaFold 3 and visualized using Schrödinger BioLuminate 2024.3.

Article Snippet: Sequencing of the chromosomal tRNA Ala(UGA) and tRNA Ala(CGA) regions was performed by first PCR amplifying the target region followed by amplicon Nanopore sequencing (Plasmidsaurus, USA).

Techniques: Sampling, Variant Assay, Infection, Titer Analysis, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Firewalled synthetic commensal blocks horizontal gene transfer in the gut

doi: 10.64898/2026.09.17.752456

Figure Lengend Snippet: ( a ) Long-term stability of Syn-Ec F 1 in the mouse gut. After gavaging n =19 animals with 10 7 CFU/animal of Syn-Ec F 1, fecal bacterial cell counts were monitored for 102 days. Plot represents geometric mean; shaded area indicates 95% CI. ( b ) Genes responsible for sulfoquinovose utilization in E. coli K-12 MG1655 and the location of mutations observed in Syn-Ec F 1 following long-term gut evolution. Green arrows indicate mutated ORFs, while magenta arrows mark genes not mutated in the evolved variant. Observed mutations are highlighted; the yihQ intergenic promoter mutation is indicated by the position of the yihQ p T-61→A and the affected yihQ . ( c ) Location of the G5→A acceptor stem mutation in the swapped-code-establishing tRNA Leu(CGA) following the long-term within-gut evolution of Syn-Ec F 1. tRNA structure was predicted using AlphaFold 3 and visualized in Schrödinger BioLuminate 2024.3. ( d ) Growth of E. coli MDS42 and the parental and gut-evolved Syn-Ec F 1 on sucrose as the sole carbon source. Bar graph shows the mean of the maximal attained OD 600 , based on n =2 independent replicates at 37 °C.

Article Snippet: Sequencing of the chromosomal tRNA Ala(UGA) and tRNA Ala(CGA) regions was performed by first PCR amplifying the target region followed by amplicon Nanopore sequencing (Plasmidsaurus, USA).

Techniques: Stability, Variant Assay, Mutagenesis