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

Abstract

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.

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

“…The pulsed-field gel electrophoresis (PFGE) analysis of chromosomes was performed in the same manner as before ( 32 ). In short, the CHEF-DR II and III Pulsed Field Electrophoresis Systems (Bio-Rad) were used for resolving DNA molecules embedded in agarose plugs.…”

“…The DNA size markers used were S. cerevisiae Chromosomes and Hansenula wingei Chromosomes (Bio-Rad). The canonical agarose gel electrophoresis analysis of chromosomes was performed using 0.5% gels and 0.5× TBE buffer in the same manner as before ( 32 ). The size marker used was Marker 3 (NIPPON GENE).…”

“…Chromosomes were purified by using NucleoBond Xtra BAC kit (Takara Bio) essentially as described before ( 32 ) but with a few modifications. Cells were harvested from 750 ml of LB cultures incubated overnight at 37°C using an INNOVA 42 (Eppendorf).…”

“…Plasmids were developed by using PrimeStar Max (Takara Bio) and KOD One Master Mix (TOYOBO) for PCR, In-Fusion (Clontech) for in vitro assembly, and chemical competent cells of HST08/Stellar cells (Takara/Clontech) for transformation. Some of the plasmids and BAC vectors were developed previously ( 32 ), and the others were newly developed by local modifications (see Table 1 ). Some of the DNA cassettes used for the λ Red recombination were developed previously ( 32 ), while the others were newly prepared.…”

“…Several strains of E. coli with two chromosomes have been established by using an additional pair of replication origins ( ori ) and partitioning loci ( par ) for the secondary chromosome ( 25 , 30–32 ). During the establishment of the two-chromosome strains of DGF-298W (2 Mb and 1 Mb) ( 32 ), we realized that chromosomes of up to 1-Mb in size can be handled in vitro as covalently closed supercoiled molecules and that 0.5-Mb supercoiled chromosomes can be even purified using a commercial DNA extraction kit ( 32 ). Furthermore, a previous study reported that up to 0.5-Mb synthetic chromosomes can be electroporated into E. coli competent cells ( 33 ).…”