Background
Plasmid instability is a significant concern in the industrial use of microorganisms. 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 compared 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 poison gene codes for a small stable protein (about 100 amino acids) whereas the antidote codes for a small unstable protein (about 90 aa) that neutralizes the poison protein. These genes are extensively used in DNA cloning technology.
Principle
In our plasmid-stability system, the antidote gene is introduced in the plasmid DNA under the control of a constitutive promoter. On one hand, the toxic gene is introduced in the chromosome of the bacteria. Expression of the poison 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 expressed/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.
Results
The system was tested in
E. coli and the results show a perfect plasmid stabilization of several vectors unstable without our technology. This technology was next applied to protein production using the T7 promoter to develop the StabyExpress™ kit. The figure below compares the production of a protein of 69 kDa with and without the StabyExpress™ technology
Higher plasmid stability = more proteins
With StabyExpress™, the plasmid is perfectly stable before and after the induction period. On the contrary, with the conventional BL21(DE3) strain, the plasmid is not stably maintained (fig. 2).
Consequently, the production of the protein of interest is higher (3 to 5 times) with StabyExpress™ (lanes c, g, and h) than with a conventional expression system with (lane f) or without (lane d) antibiotics. Lanes a, b, and e are uninduced controls. Note that no over-production of the antidote is detectable (fig. figure below).
Legal notices
The StabyExpress™ kit is covered by worldwide patents. The kit is sold under a license from the
Université Libre de Bruxelles (Belgium). The kit is sold for research purpose only. A license
from Delphi Genetics SA is required for any commercial use (Please, contact Delphi Genetics SA
at
delphigenetics@delphigenetics.com).