Team:University College London/Module 6

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. Horizontal gene transfer can 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 nuclease. Any DNA released from the cell would therefore be digested, before it could diffuse, and transform wild-type bacteria. For this system we have selected a extracellular nuclease from Staphylococcus aureus (nucB), which has been well characterized (1-3) however it lacks the signal peptide for secretion to periplasm in E. coli. We chose DsbA (BBa_K243002) signal sequence that enables export of our nuclease to periplasm, thus it will allow us to digest extracellular genetic material.

As horizontal gene transfer can also occur via bacteria conjugation, 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.

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.

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 separate systems is far more robust as it will provide reinforcement if a single system is knocked out. (Ronchel et al. 2001)

Reference

1. Okabayashi K, Mizuno D. (1974) Surface-bound nuclease of Stapylococcus aureus: purification and properties of the enzymes. Bacteriol. 117(1):222-6

2. Suciu D, Inouye M. (1996) The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli. Mol Microbiol. 21(1):181-95

3. Cooke GD, Cranenburgh RM, Hanak JA, Ward JM. (2003) A modified Escherichia coli protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid. J Biotechnol. 101(3):229-39.