Dik, D. A. | Search bioRxiv

bioRxiv2024

Natural Selection in a Synthetic Yeast Endosymbiont Promotes Stable Coexistence

Bacteria engulfment by a higher order host is believed to be the beginning of an evolutionary process that ultimately formed mitochondria. In an effort to experimentally elucidate the early effects of natural selection on bacteria resident in a eukaryotic host, a synthetic endosymbiont model system has been exploited. Here we describe a reproducible series of mutations that were observed after Escherichia coli was passaged within Saccharomyces cerevisiae for >8 passages which led to enhanced coexistence of bacteria within the yeast. These naturally selected mutations, formed by gene acquisition of trans-posable elements in rcsC, cpxA, and idnK, result in both functional and non-functional protein products with phenotypic effects on the bacteria that promote endosymbiont stability.

synthetic biology↗

bioRxiv2024

An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 x 10-5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 {beta}-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.

synthetic biology↗