Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation.

ABSTRACT: The physical stability of bacterial chromosomes is important for their in vitro manipulation, while genetic stability is important in vivo. However, extracted naked chromosomes in the open circular form are fragile due to nicks and gaps. Using a nick/gap repair and negative supercoiling reaction (named SCR), we first achieved the negative supercoiling of the whole genomes extracted from Escherichia coli and Vibrio natriegens cells. Supercoiled chromosomes of 0.2-4.6 megabase (Mb) were separated by size using a conventional agarose gel electrophoresis and served as DNA size markers. We also achieved the enzymatic replication of 1-2 Mb chromosomes using the reconstituted E. coli replication-cycle reaction (RCR). Electroporation-ready 1 Mb chromosomes were prepared by a m

SUBMITTER: Fujita H 

PROVIDER: S-EPMC9486964 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature