Safety in the Environment

To dramatically decrease the chance for horizontal gene transfer we have implemented our "Triple Lock” system:

1. Colicin E2 DNA Degradation Switch - Colicin E2 is a nicking endonuclease which we have implemented into a timed kill switch that will degrade any DNA in the maxicell immediately prior to the end of its active metabolic timeframe. This significantly reduces the chance of horizontal gene transfer. The kill switch genes would be recoded as described in our semantic containment model system.
2. Semantic Containment - This system prevents any environmental cell that may obtain a gene from a maxicell instructor plasmid (by any mechanism of HGT) from reading or expressing that gene. Genes on the instructor plasmid are recoded by substitution of serine codons for amber STOP codons. Our probability model for semantic containment predicts probability of gene expression with increasing numbers of amber stop codons in a coding sequence.
3. Triclosan Resistance - The spread of antibiotic resistance is a major problem, one that we did not want to add to. We have therefore replaced the antibiotic resistance gene on our instructor plasmid with a gene for resistance to the biocide, Triclosan (FabV). Triclosan is now rarely used in any health or beauty products, and therefore resistance would confer no competitive advantage to an environmental cell or pose any health danger to humans.

Maxicells are naturally safer in the environment due to their inability to reproduce - meaning they cannot accumulate mutations over successive generations and evolve out of our control.

Lab Safety

The parts/gene products we have used have no effect on humans. We used only non-pathogenic well characterised lab strains for our experiments. Some protocols required the use of UV light (the UV maxicell protocol and viewing DNA in a polyacrylamide gel). All necessary precautions were taken to prevent harm (tinted goggles/ viewports and black boxes with automatic shut offs when opened). The necessary risk assessments were completed and approved for each stage and element of our project and experimental design. Wore personal protective equipment when necessary. Used fume hoods when working with chemicals which created hazardous fumes.

Future Proofing and GM Law

Because we have made a chassis for environmental release we were concerned that (although we haven’t made it physically easier to release GM into the environment) we may have made it easier to release GM legally and we wanted to make sure that GM law was robust enough to prevent any unsafe uses of our chassis (and hoped it gave safe uses a chance). For this reason we filled out a GM Environmental Release Application form as if we were to release an arsenic biosensor in our chassis and we spoke to the GM Inspectorate.

Future Work for Biosafety

Before potential release to the environment, the effect of transposons on the mobility and HGT rate of genes in the instructor plasmid needs to be determined if the maxicells are to be made from strains with transposons.

The effect of phages on HGT in maxicells also needs to be determined.

Tests in a mock “wild” environment (e.g. soil in the lab) should be carried out to determine if resistance could be spread from maxicells.

All future work to determine biosafety should take into consideration the far larger scale of earth as opposed to lab experiments. Future work and the politics surrounding it needs to consider how safe is safe enough as the potential benefits of being able to use GMOs more freely in the environment are massive but safety can never be 100% guaranteed in anything no matter how close you get.

Semantic containment - work with a RF1 knockout strain that has all amber stop codons removed from the chromosome.

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