Team:UT-Knoxville/Proof
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Team:UT-Knoxville/Team
UTK
iGEM
Bronze Medal
- We registered our team and are excited to attend the Giant Jamboree in Boston.
- All deliverable requirements have been met.
- Our attributions are placed on the tab “Attributions->Group” of our wiki (click here).
- Characterization/ Contribution (Interlab study):
- We added the following parts to the registry: BBa_K2451002, BBa_K2451004, BBa_K2451006, BBa_K2451008, BBa_K2451013, and BBa_K2451015.
- Interlab Study: Our team participated in the Interlab study. Although the strains did not express GFP, it was deemed acceptable by HQ.
Silver Medal
- Validate Part: BBa_K2451013 (UTK03) showed inducible GFP expression in the presence of m-xylenes. Click here to read more about this construct.
- Collaboration: Team UT-Knoxville has struggled with recruiting new members, fundraising, and troubleshooting. This year, we looked to Team Aachen for mentorship. For details on our collaboration with Team Aachen, click here.
- Human Practices: We worked with various institutions to organize multiple outreach events in the Knoxville area. Details can be found here.
Gold Medal
- Integrated Human Practices:
- Improve a Previous Part:
- Demonstrate your Work:
Through testing our bacteria with gasoline last year, we hoped to one day be able to implement our engineered cells directly onto an oil spill and be able to watch our cells degrade the harmful oil from the environment. To better understand what this would entail, we interviewed a professional in the field, Dr. Terry Hazen who uses microbial ecology and environmental engineering to research bioremediation and oil recovery.
While interviewing Dr. Hazen, he explained how we would approach our project in regard to implementation, as well as general tips surrounding permits, patents, and other projects dealing with hydrocarbon degradation. He discussed the several steps to implement a project like ours, and instead, encouraged us to pursue creating a bioreactor that directly connects to the oil processing pipeline producing the BTX we convert. This way, we can quickly implement our project in a real-world setting, and avoid repercussions from releasing our cells into the environment.
Taking his advice, we sought modifications to our bioremediation organism that would help its function in a bioreactor setting, as opposed to modifications that would allow it to survive in the environment, and are now making moves to design a bioreactor next year that would expand our results from previous years and allow us to implement our project in a real-world setting. This will in turn be more realistic than releasing our bacteria into the environment like we had planned to pursue. We are thankful for the applicable advice that we received while interviewing an expert in the field of bioremediation, and we hope to continue discussion of our project’s future with Dr. Hazen in the future.
Our problems with 2013 IGEM Peking’s Pu promoter (BBa_K1031803) were twofold. First, it had to be coupled with a separate plasmid to accommodate xylR, the regulatory activator. Second, our team was unable to reproduce m-xylene induction using their circuit. Our team improved on this part by establishing a single plasmid for Pu regulation (coupled xylR and Pu onto the same vector). Additionally, we improved translation rates by adding untranslated regions (UTRs) between ribosome binding sites and start codons. Future teams may now BioBrick our Pu-GFP biosensor (BBa_K2451013), or Gibson another structural gene in the place of GFP.
With 0.1 mM IPTG and 1 mM m-xylenes added to our BBa_K2451013 construct, our Pu promoter showed ~4x GFP expression than our solely 0.1 mM IPTG construct. Thus, our regulatory sequence including xylR regulatory protein and Pu promoter was able to significantly change expression levels based on the presence of aromatic compounds. These results are reflected on Figures 2 and 3 on the Project page.
1 mM m-xylene induction of BBa_K2451015 (UTK05) revealed conversion of m-xylene to 3-methyl benzyl alcohol and m-tolualdehyde while blank plasmid, BL21(DE3), and the construct without m-xylenes did not display peaks signifying either the alcohol or aldehyde presence. Our catalytic genes were therefore able to transcribe and produce functional proteins to convert our aromatics under our Pu promoter. This would allow for fine-tuned protein expression to convert aromatics to aromatic aldehydes in our project’s desired bioreactor setting. The results described above are shown in Figures 6 and 7 of the Project page.
