Module 6: Containment

Description

As our project suggests the release of genetically modified bacterium into the environment, we feel it is necessary to contain the risk of spreading genetic information by horizontal gene transfer. To do so we are suggesting a trio of protective systems – consisting of two toxin/anti-toxin pairs - Colicin-E3/Colicin Immunity E3 and Holin/Anti-Holin endolysin– and an excreted nuclease.

Having such a system in place will prevent 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 HGT 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 Ramos 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 antitoxin. Production of toxin in the absence of antitoxin leads rapidly to cell death, which should prevent the sharing of genetic information.

Colicin E3 is an antibiotic protein which has ribonuclease activity that targets the 16S ribosomal RNA. This fatally impacts a cells ability to synthesise proteins, unless the antitoxin Im3 is also present. Im3 is called an immunity protein – so named because it binds colicin E3 to prevent the ribonuclease activity. In our bacterial system, Colicin E3 will be present on a plasmid, and IM3 in the genomic DNA, so that both are constitutively produced. During conjugation, the conjugating partner will replicate, translate, and transcribe the Colicin E3 gene leading to cell death.

This will be reinforced by another toxin/antitoxin system – a holin/antiholin endolysin pair. As before, the toxin holin will be carried by a plasmid and driven to constant expression by the constitutive promoter BBa_J23119. Holin causes disruption of the inner cellular membrane, such that it allows an enzyme – endolysin – to enter the periplasm. Endolysin attacks the peptidoglycans of the periplasm, leading to cell lysis. Inhibition of this system can be achieved by having antiholin, which binds holin to prevent the disruption of the cell membrane, constitutively expressed in the genomic DNA. As before, conjugation with other bacteria will lead to replication of the damaging holin and endolysin genes, but not the rescue protein antiholin. All conjugating partners will suffer cell lysis as a result.

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 an extracellular 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