Team:WLC-Milwaukee/Safety

Safety

Safety was of paramount importance to our team this year. The first safety priority while doing our work was the preservation of ourselves and teammates. To ensure correct laboratory precautions within the lab, every member of our team was required to take CITI lab training, which alerted us to the potential dangers of lab work while providing preventative measures and techniques for staying safe in the lab. After having completed these certifications, all members of the team were required to go over basic lab techniques such as pipetting.

Once the safety of our team was assured, we were able to begin thinking about the safety of our product in both the envirionment and the organisms. First, we decided to place our plasmid in Escherichia coli, more specifically E. coli Nissle 1917. This specific strain was chosen due to its probiotic nature. E. coli Nissle 1917 has been used medicinally since 1917, thus the use of our modified strain would not be introducing a new killer strain of bacteria. Many tests have also been performed on this strain of E. coli, none of which showed any signs of toxins; rather they showed “gene clusters responsible for synthesis of several so-called ‘fitness factors’ which contribute to the strain’s probiotic nature.”[1]

With a safe host cell determined, our team then began to think of methods for shutting down our plasmid should an unforeseen issue arise. To do this we decided to integrate a kill switch into our system. We chose the Tse2 toxin which is created through the expression of the T5 Cumate promoter. This promoter is inhibited by the presence of the CymR protein, which is regulated by p-Cumate. Thus, if our plasmid mutated out of control we simply need to expose the cells to p-Cumate to deactivate the CymR protein and to begin production of the Tse2 toxin.

While our team did spend resources trying to integrate a successful kill switch in case the inevitable unforeseeable event were to occur, we realize that kill switches are not completely infallible. Mutations constantly occur in bacteria. Due to these constant mutations, the kill switch coding sequence will eventually have a mutation which will make the plasmid uncontrollable. Therefore, the Sugar Rush team, while unable to encorporate the following safety mechanisms, has considered these other means of ensuring the safety of our project.

First, we considered simply adding a second kill switch. This addition would drastically reduce the chances that a mutation would occur and cause problems. This is because both genes are far less likely to be mutated coincidentally within a single organism. Next we considered adding gene flow barriers to prevent conjugation amongst different bacteria. "Gene-flow barriers are created by including a killer gene in the rDNA and placing the rDNA into an immune host. Immunity from the killer gene is provided by a repressor protein that blocks killer gene expression. If unintended hosts take up the engineered DNA, the lethal gene is decoupled from immunity and the new host cell dies.”[2] The final safety mechanism which could be attempted in the future would be to create Auxotrophic cells. This means making cells which are dependant upon something within the target organism (in our case a cow) to live. [2] If the cells were ever to escape the organism they would die and not be able to spread due to this organism-specific dependency. [1]