Safety in the Environment

To dramatically decrease the chance for HGT we have implemented a “triple lock” system:

1. Colicin E2 DNA Degradation Switch - Colicin E2 is a DNase which we have implemented into a “timed” kill switch which will degrade any DNA in the maxicell before maxicell’s metabolic timeframe end. This will significantly reduce the chance of HGT and the kill switch genes would be recoded as described in semantic containment.
2. Semantic Containment - The recoding of genes on the instructor plasmid and the lack of chromosomal DNA prevents cells in the environment from being able to use any of the genes on the instructor plasmid therefore they cannot give a wild cell an advantage over others in the event of HGT from the maxicell to the wild cell. Any gene given to the instructor plasmid would be recoded for semantic containment.
3. Triclosan Resistance - The spread of antibiotic resistance is a major problem, one that we didn’t want to add to. Instead of antibiotic resistance we use triclosan resistance in our chassis and in far lower amounts than found in toothpaste and industrial outflow. There are many benefits to using triclosan including increased environmental safety over antibiotics.

In addition to the work we have done to improve the chassis’ biosafety, maxicells are naturally safer in the environment due to their inability to reproduce which means they can’t accumulate mutations over generations and thus can’t 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 protocells 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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