Rapid genome recoding by iterative recombineering of synthetic DNA

Abstract

Genome recoding will provide a deeper understanding of genetics and transform biotechnology. We bypass the reliance of previous genome recoding methods on site-specific enzymes and demonstrate a rapid recombineering based strategy for writing genomes by Stepwise Integration of Rolling Circle Amplified Segments (SIRCAS). We installed the largest number of codon substitutions in a single organism yet published, creating a strain of Salmonella typhimurium with 1557 leucine codon changes across 200 kb of the genome.

Enabling technology and core theory of synthetic biology

“…In addition to Saccharomyces cerevisiae, Bacillus subtilis, Salmonella typhimurium, and E. coli are three alternative hosts for in vivo genome assembly (Fredens et al, 2019;Itaya et al, 2008;Itaya et al, 2005;Lau et al, 2017). A 3.5 Mb genome has been assembled into a B. subtilis genome by the "inchworm" method (Itaya et al, 2005).…”

“…Using a B. subtilis genome (BGM) vector, a 16.3-kb mouse mitochondrial genome and a 134.5-kb rice chloroplast genome were successfully integrated by homologous recombination into a B. subtilis genome (Itaya et al, 2008). A 200 kb S. typhimurium segment was replaced by synthetic DNA through a process called stepwise integration of rolling circle amplified fragments (SIRCAS) (Lau et al, 2017). In E. coli, a conjugationbased strategy, coupled with repetitive replicon execution for enhanced genome engineering through programmed recombination (REXER), enabled the synthesis of a ~4 Mb recoding genome (Fredens et al, 2019;Wang et al, 2016).…”