Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes

Abstract

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.

Synthetic chromosomes, genomes, viruses, and cells

“…There could be commercial entities that-without publicizing their work-constructed or are constructing bacteria, yeasts, or algae with synthetic genomes to produce molecules that could not be made otherwise. On the other hand, there have been numerous publications describing computational approaches to design recoded and minimal genomes as well as laboratory approaches that may facilitate grand scale genome reconstruction and methods to replace large sections of native bacterial genomes with synthetic versions (Krishnakumar et al, 2014;Kuznetsov et al, 2017;Lamoureux et al, 2020;Lau et al, 2017;Libicher et al, 2020;Rees-Garbutt et al, 2020;Yoneji et al, 2021).…”

Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation

“…Recently, we succeeded in the transformation of Escherichia coli via electroporation with a 1.0 Mb chromosome purified using a bacterial artificial chromosome (BAC) purification kit. 9 A population of negatively supercoiled 1 Mb chromosomes survived the purification and electroporation processes, probably owing to the compacted structure. 10 Furthermore, these supercoiled chromosomes were separated by size using conventional agarose gel electrophoresis.…”

“… 10 Furthermore, these supercoiled chromosomes were separated by size using conventional agarose gel electrophoresis. 9 These findings suggested that even larger chromosomes might be manipulatable when covalently closed and negatively supercoiled.…”

“…Among the three split-chromosomes (1.12, 0.84, and 1.02 Mb), the second and third ones were individually introduced via electroporation into an E. coli cloning strain HST08 as an extra chromosome. 9 This meant that the two chromosomes are “portable.” In contrast, the first one was not portable, probably due to the burden of the duplication of the genomic core region or due to the lack of any additional chromosome partitioning system and segregation system. 9 In this study, we show that enzymatically supercoiled bacterial chromosomes can be handled in vitro and are useful for genome analysis and chromosome implantation.…”

“… 9 This meant that the two chromosomes are “portable.” In contrast, the first one was not portable, probably due to the burden of the duplication of the genomic core region or due to the lack of any additional chromosome partitioning system and segregation system. 9 In this study, we show that enzymatically supercoiled bacterial chromosomes can be handled in vitro and are useful for genome analysis and chromosome implantation. Also, we show that the genetic stability of chromosomes can be improved according to a portable chromosome format.…”