Preliminary Design Considerations

The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. Despite our belief that the metabolic burden of this system on our bacteria would not allow them to outcompete any native organisms, as we detail in our interviews page, our dialogue with experts really emphasized the need to design such a system so as to minimize any escape of our bacteria regardless. As such, we designed a closed biosensor and a closed bioreactor which incorporated built-in structural design safety mechanisms. In order to implement one more level of control, which industry felt was needed, we wanted an additional genetic-based containment mechanism to kill our bacteria upon escape from our system, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We implemented novel riobo-killswitch parts. These contain riboswitch regulatory elements and exo/endonuclease kill genes.

Design Challenges from Industry

As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make informed design choices so as to maximize the probability of our system actually being implemented one day.

Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.

Our Solution

With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10^-6 per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor design Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms.

As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry conerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.