Bacterial strains with improved plasmid stability
The present invention relates to the propagation of covalently closed circular recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly is strain modifications that improve strain viability, plasmid stability, plasmid production yield, and plasmid-directed protein production yield, using said DNA molecules in fermentation culture.
1. A method for increasing production yield of covalently closed super-coiled plasmid DNA containing a target gene, comprising the steps of:
a. cloning part or all of the target gene into a RNA expression vector, the RNA expression vector expressing a sense or an antisense RNA of the target gene;
b. integrating a single copy of the RNA expression vector into the E. coli bacterial genome to create a modified E. coli bacterial cell line;
c. transforming the plasmid into the modified E. coli bacterial cell line to form resultant transformed bacterial cells, wherein the modified E. coli contains a single chromosomal copy of the sense or the antisense RNA of the target gene acting as a regulator to generate sense or antisense RNA to effectively downregulate translation of multiple plasmid copies of the target gene, the modified E. coli bacterial cell line being competent for transformation; and
d. isolating the resultant transformed bacterial cells to create a seed stock for plasmid propagation;
whereby the modified E. coli bacterial cell line improves the plasmid copy number in subsequent shake flask and or fermentation culture.
2. The method of claim 1 wherein the plasmid is a eukaryotic expression vector.
3. The method of claim 1 wherein the plasmid is a prokaryotic expression vector.
4. The method of claim 1 wherein the RNA expression vector expresses an antisense RNA of the target gene.
5. The method of claim 1 wherein the RNA expression vector expresses a sense RNA of the target gene.