Team:Pitt/Competition/Judging

Bronze Medal

1. Register team.
2. Complete judging form.
3. Team Wiki.
4. Present a poster and talk at the iGEM Jamboree
5. Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry

Five biobrick parts were constructed and submitted, two of which are novel parts: 1) hsp60 promoter from M. bovis expected to work in P. acnes (BBa_K1548000), and 2) hsp60 promoter + TM4 bacteriophage RBS bacteriophage, also expected to function in P. acnes (BBa_K1548001). The additional parts are 3) a protein generator system using the hsp60 promoter to produce mRFP1 (BBa_K1548002), 4) a protein generator system using the hsp60 promoter to produce the blue light regulator to lead to blue light induction of mRFP1 (BBa_K1548003), and 5) a composite part (BBa_K1548004). The new parts are documented in the registry along with citations for the sequences.

Silver Medal

1. Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected.
2. Document the characterization of this part in the “Main Page” section of that Part’s/Device’s Registry entry.
3. Submit this new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines).

M. bovis hsp60 was characterized in E. coli attempting to express mRFP1 from the promoter and a strong E. coli RBS, B0034 (BBa_K1548002). An induction experiment was then performed to determine whether the hsp60 promoter would be active in E. coli by growing Mach1 E. coli cells containing this plasmid in LB + Cam overnight. The results documented on the part page show that we observed no activity of the hsp60 promoter in E. coli. Consequently, in silico E. coli promoter prediction analysis was performed on the hsp60 promoter using the BPROM program in the Softberry software package. This software predicted that the promoter would not function in E. coli, and thus our negative results in E. coli were validated. Promoter function in P. acnes is still predicted, however, because P. acnes is more closely related to M. Bovis, belonging to the same phylum.

4. Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project.

The chassis developed by the 2014 Pitt iGEM Team naturally grows on human skin, therefore there are a number of potential safety risks involved in engineering and working with this bacterium. While none of the genes presented are toxic nor are expected to lead to any adverse effects, and given how engineered strains typically experience a reduction in fitness compared to non-engineered strains, some safety precautions need be considered due to the direct contact of the organism with humans. Additionally, the engineered bacteria would also contain resistance to erythromycin, because the gene vectors were selected for on antibiotic plates, therefore the chassis has an additional, albeit remote, possibility for horizontal gene transfer. Thus, the ideal implementation of the system includes a kill switch to efficiently kill off any engineered bacteria in the event of a negative reaction to the engineered bacteria. Several such systems have been developed by previous iGEM teams. Another possible solution would be the creation of a genetic fence such that the bacteria would only survive in the presence of some exogenous inducer on the skin. The fence would be a substance topically applied to the skin, forcing the engineered bacteria to only inhabit the “fenced” skin area. As P. acnes traditionally inhabits the skin follicle, the substance would have to penetrate to this level. It is likely that either of these systems would provide all of the necessary safety precautions to lead to removal.

Gold Medal

1. Help any registered iGEM team from another school or institution by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.

As outlined under the iGEM Collaborations section, we made significant efforts to aid other iGEM teams, most notably Stony Brook iGEM and Montgomery High School iGEM, who were experiencing the growing pains of a first-year team. In-person meetings were held with Stony Brook and Montgomery to offer advice on laying the foundations, gathering support, and troubleshooting in the lab. These discussions also led to the creation of a Pitt iGEM Wordpress, where a list of various how-to articles is maintained for other iGEM teams struggling with similar problems. We also participated in the University of Virginia’s survey and the Pittsburgh area team meetup co-hosted by Carnegie Mellon iGEM, along with various community outreach events with Carnegie Mellon iGEM.

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