Background
Plasmid instability is a significant concern in protein production. Typically, protein-production processes require the use of bacterial plasmids as vectors carrying the gene to be over-expressed. It has been demonstrated that the growth rate of plasmid-bearing cells is significantly reduced relative to that of a plasmid-free host, simply because protein production (corresponding to the gene-of-interest over-expression) represents a significant burden on cellular metabolism. Antibiotic-resistance genes are the most common selectable markers used in fermentation procedures to avoid plasmid-free cells to survive and dominate the culture. However, contamination of the product or biomass by antibiotics (or genes encoding an antibiotic resistance) is unacceptable from a medical or regulatory perspective (cf. FDA recommendations). Our new stabilization system is based on the use of antidote/poison genes naturally found in plasmids, chromosomes, and bacteriophages. The ccdB selection gene codes for a small stable protein (about 100 amino acids) whereas the ccdA antidote gene codes for a small unstable protein (about 90 aa) that neutralizes the selection protein. These genes are extensively used in DNA cloning technology and are only active in enterobacteriacae (E. coli, Salmonella).

Principle

In our plasmid-stability system, the antidote gene is introduced in the plasmid DNA under the control of a constitutive promoter. On the other hand, the selection gene is introduced in the chromosome of the bacteria. Expression of this selection gene is under the control of a promoter strongly repressed by the antidote protein. Hence, when the plasmid is present in the bacteria, the poison is not produced. On the other hand, when the plasmid is lost, the antidote is degraded and the production of the toxin is induced, causing cell death.

The StabyExpress system is compatible with the use of an auto-inducible medium as Staby^TMSwitch.