Conjugation-based genome engineering in Deinococcus radiodurans

Abstract

D. radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based transformation system from E. coli to D. radiodurans allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by target-ing restriction-modification system genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facili-tate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications.

Microbial applications for sustainable space exploration beyond low Earth orbit

“…Conjugation has been demonstrated within and between many species of Gramnegative and Gram-positive bacteria in a laboratory setting, and from bacteria to eukaryotic cells. Conjugative tool development for many of these species could have implications for their use in the space environment including those developed to target opportunistic pathogens (e.g., Enterococcus, Listeria, Staphylococcus 449 , Streptomyces 450 , and S. enterica 451 ), or those that could be used to engineer useful strains such as probiotic candidates (e.g., Bacillus 449 , Bifidobacterium 452 and Lactobacillus 453 ) or extremophilic microbes (e.g., D. radiodurans 454 , yeast 455 or algae 267,268 ). When coupled with CRISPR/Cas technologies conjugation can be used for the modulation of microbiomes and as a method for targeted eradication of human pathogens on Earth.…”

A Survey on Immune System in Genetic Engineering and their Applications

“…The majority of time, this DNA is present in the form of plasmid. This DNA is directly transferable and it requires a direct contact between the donor cells and recipient cells by including a conjugation tube [13].…”