https://www.selleckchem.com/products/cpi-203.html 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 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.Background Cardiac sympathetic denervation (CSD) is being used in the management of refractory ventricular tachycardia and electrical storm. However, data on the role of CSD in the management of ventricular arrhythmia is limit