Foo, G. W. | bioRxiv Science

bioRxiv2026

Clinical Campylobacter jejuni isolates: genomes and genetic tools

Campylobacter jejuni is a major cause of food-borne gastroenteritis and is responsible for substantial mortality and economic losses in meat and dairy production. Detecting C. jejuni in contaminated food samples remains difficult because current assays are culture-based, slow, and can yield false positives. As a result, contamination may not be identified for several days, limiting detection at the point of production. Developing improved assays has also been challenging because Campylobacter genetics and the biology of clinical isolates remain poorly understood. Here, we expand the C. jejuni genetic toolbox by sequencing two strains, HC1 and RM1164, derived from patient and food samples. We identified two cryptic plasmids in HC1, one potentially capable of conjugation and another conferring tetracycline resistance. We also engineered a mobilizable plasmid carrying an OriT sequence that can be transferred from Escherichia coli donor strains to C. jejuni RM1164 by conjugation. Together, these clinical isolates and the plasmid system expand the genetic tools available for C. jejuni.

synthetic biology↗

bioRxiv2023

Intern-based thermoregulated meganucleases for biocontainment of genetic material

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18{degrees}C and not at higher temperatures. We constructed an orthogonal set of temperature sensitive-meganucleases, or TSMs, by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18{degrees}C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. We demonstrated that TSMs eliminated plasmids from the E. coli Nissle 1917 strain after passage through the mouse gut when fecal resuspensions were incubated at 18{degrees}C but not at 37{degrees}C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

synthetic biology↗