Module 6: Containment

Description | Design | Construction | Characterisation | Modelling | Results | Conclusions

Description

As our project suggests the release of genetically modified bacterium into the environment, we feel it is necessary to contain the risk of horizontal gene transfer. To do so we are proposing a multi-containment system, consisting of three toxin/anti-toxin pairs - Holin / Anti-Holin Endolysin, Colicin-E3 / Colicin Immunity E3, and Endunuclease EcoRI / Methyltransferase EcoRI. Our system will also have an exonuclease component.

Having such a system in place will prevent the transfer of synthetic genes into wild type bacteria; minimising the interference of our bacteria with the natural environment. Having more than one system is essential – even a system with 100% success at preventing horizontal gene transfer can be diminished by spontaneous mutations in the gene sequence. The use of three separate systems is far more robust as it will provide reinforcement if a single system is knocked out. (Ronchel et al. 2001)

The system will be driven by a constitutive promoter (BBa_J23119), to ensure that the release of genetic information is constantly neutralised.

Toxins will be carried on a plasmid and the antitoxin on the genomic DNA. As conjugation leads to the sharing of plasmid DNA, but not genomic DNA, the conjugating partner to our bacteria will receive the gene encoding the toxin, but not the related anti-toxin. Production of toxin in the absence of anti-toxin leads rapidly to cell death, which should prevent the sharing of genetic information.

However, HGT can also occur by the release of genetic information through cell lysis, and subsequent transformation of other bacteria.

We decided we could target this system by constitutively expressing a periplasmic nuclease. Any DNA released from the cell would therefore be digested, before it could diffuse. For this system we have selected a nuclease from Staphylococcus aureus.