Open AccessConjugation-Based Genome Engineering in Deinococcus radiodurans
Author(s) -
Stephanie L. Brumwell,
Katherine D. Van Belois,
Daniel J. Giguere,
David R. Edgell,
Bogumil J. Karas
Publication year - 2022
Publication title -
acs synthetic biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.156
H-Index - 66
ISSN - 2161-5063
DOI - 10.1021/acssynbio.1c00524
Subject(s) - deinococcus radiodurans , plasmid , genome , genome engineering , biology , synthetic biology , genetics , computational biology , genome editing , gene
Deinococcus 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 DNA transfer method 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 targeting restriction-modification 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 facilitate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications.