Journal
NUCLEIC ACIDS RESEARCH
Volume 49, Issue 15, Pages 8407-8418Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab298
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Funding
- Japan Science and Technology Agency (JST) [JPMJCR18S6]
- Japan Science and Technology Agency
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This study developed a method to split the genome-reduced E. coli into three 1 Mb chromosomes, which allows for further genetic engineering of tripartite-genome cells with minimal impact on growth rate. Additionally, the purified 1 Mb chromosomes can be successfully electroporated into other cells under experimental conditions.
In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.
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