Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation

[Image: see text] 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 modified SCR performed at a low salt concentration (L-SCR) and directly introduced into commercial electrocompetent E. coli cells. Since successful electroporation relies on the genetic stability of a chromosome in cells, genetically stable 1 Mb chromosomes were developed according to a portable chromosome format (PCF). Using physically and genetically stabilized chromosomes, the democratization of genome synthetic biology will be greatly accelerated.