Irving, O. J. | bioRxiv Science

bioRxiv2025

A modified methyl transferase cofactor to selectively disable gene expression in E. coli

Artificial control of gene expression in bacteria offers interesting prospects for influencing bacterial pathogenicity and antibiotic resistance. We show that the methyl-transferase cofactor, AdoHcy azide, can disable gene expression in modified plasmids in some strains of E. coli, where ampicillin and kanamycin resistance as well as eGFP genes were selectively and independently disabled. The disabling of transcription is likely due to steric inhibition during transcription initiation, which is further confirmed by Sanger and nanopore sequencing results. Both sequencing methods showed that 3-6 nucleotides were absent from around the modification site, with the post growth, extracted AmpR/ eGFP plasmid showing evidence of restriction, with sections of the plasmid, including the modification site, missing for the AdoHcy azide modified plasmids. Notably, the AdoHcy azide modification on the DNA is resistant against demethylation in the BL21 strain of E. coli.

genomics↗

bioRxiv2023

Sterically enhanced control of enzyme-assisted DNA assembly

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA, which spatially restrict enzymatic activity. This approach, Sterically Controlled Nuclease Enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows for the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, using the same starting materials conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation.

biochemistry↗