Team:Baltimore BioCrew/Improve

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Improve A Part

• HKUST Killswitch






• Our Killswitch

HKUST Killswitch

Overview and Experimentation

The HKUST iGEM team designed a killswitch in 2012, part BBa_K733012. This part was designed to release a growth inhibiting endotoxin when in the presence of xylose. According to their wiki and registry page, the part worked as intended. HKUST tested their killswitch by measuring turbididty. They innoculated their bacteria in solutions containing varying amounts of xylose (0% to 40% at increments of 5%). They were put in a shaking incubator for 16 hours and then measured. As intended, the levels went down as the xylose concentration increased.

Our findings

When we ligated and transformed BBa_k733012, we had positive results for colonies. However, after replicating their experiment exactly, we got inconsistent data. We attempted to eliminate any possible variables, running the experiement multiple times to minimize chances of contamination, use different medias, allow the cells to grow for varying amounts of times, and raised the xylose levels. Every time, we got inconsistent data. Seeing as we had accounted for nearly every foreseeable issue, we came to the following conclusion: either HKUST's data was not able to be replicated, or the entire sample of DNA in our well plate had been contaminated.

Our Killswitch

We wanted our killswitch to be safer and have a more guaranteed death, so we decided to make the endotoxin release when deprived of xylose, as opposed to to in the presence of it. As our part would be in the body, it was imperative that we could ensure cell death exactly when we want it. On top of that change, we also used a new terminator, part BBa_B1002, a well characterized and simpler terminator. We used this for two reasons. One was that we had higher confidence that it would work, due to its wide scale usage in iGEM. Secondly, the initial construct was too complex to be synthesized accurately, so a smaller and simpler terminator made it easier.

Experimentation

After we had established a colony of our killswitch, we proceeded to run another experiment to determine whether or not our construct was successful.