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

Although 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 strain was split into two chromosomes (2-Mb and 1-Mb), of which smaller one has the origin of replication and the partitioning locus of the 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 (-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 strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable by using the reconstituted chromosome replication cycle reaction (RCR). These strategies and technologies would make popular cells a productive factory for designer chromosome engineering.