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

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.

Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli.

Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which the smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision, which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new cloning method (oriT-POP cloning) and a fully equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.