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