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Team:ULB-Brussels/Safety
From 2009.igem.org
Revision as of 11:28, 21 October 2009
In this work, we were aware of some ethical issues, in particular the use of antibiotics. Antibiotics are used to select all types of plasmidic vectors in bacteria which is a significant concern in industrial cultures producing recombinant proteins or DNA. As plasmid-free cells growing faster than plasmid-carrying cells, the yield and the production reproducibility of recombinant molecules are significantly lowered in the absence of plasmid selection by the appropriate antibiotic. To overcome the plasmid instability, many antibiotic resistance genes are used as selectable markers in fermentation processes. Antibiotics are expensive and they pose safety problems: they contaminate the production product. The Belgian company Delphi Genetics has developed a strategy called “StabyTM system” which relies on the use of poison-antidote genes.
The StabyTM system is based on the poison-antidote ccd module. Poison-antidote modules are found in natural plasmid in which they serve to the plasmid maintenance. The CcdB protein (poison) is cytotoxic and poisons DNA-gyrase complexes. Expression of this gene in the absence of its cognate ccdA antidote leads the death of the bacteria. The product of the ccdA gene (antidote) antagonizes this toxic activity by forming a poison-antidote protein complex. If a plasmid carrying the ccd module is lost at cell division, the plasmid-free bacteria contain poison and antidote proteins in their cytoplasm. Since the antidote is unstable and degraded by a host protease, the poison will be free and able to poison DNA-gyrase complexes. This will lead eventually to cell death.
In the StabyTM system, the antidote gene (ccdA) is introduced in the plasmid DNA under the control of a weak constitutive promoter: the mob gene promoter, which originates from a broad host range plasmid (pBHR1). On the other hand, the toxic gene (ccdB) is introduced in the E. coli chromosome of the bacteria. Expression of the poison gene is under the control of a promoter strongly repressed in the presence of the plasmid. Practically, 100% of the bacteria will carry the vector. If they lose the vector, they will not obtain a growth advantage, but will die. Upon induction, all the bacteria will start producing the recombinant protein. It will lead to higher yields of the recombinant protein and less background caused by unwanted proteins. Therefore, higher plasmid stability means higher protein of interest production.
We think it could be relevant to implement this antibiotic free expression strategy in our project. This is the reason why we designed 2 bricks containing the ccdA gene. In order to carry out transformation, bacteria should be ordered with the ccdB gene in their genome from Delphi Genetics.
