Data Page

Characterization of New Parts Submitted to the Registry

• Submitted two new reporters to the registry: Beta-lactamase (Bba_ K1189008) and Recombinant Human Ferritin (Bba_ K1189008). We submitted these parts alone, with 6-his tags and lacI inducible promoters and fused to TALE-A (Slovenia 2012). In addition, we submitted Recombinant Human Ferritin with E coil dimerization domains in order to be used as a modular scaffold (Bba_K1189018).
• Expressed Ferritin (Bba_ K1189008), which possesses 24 subunits, and showed successful chemical conversion of our expressed protein into Prussian Blue ferritin. We also showed that this part could feasibly be used as a reporter in a nitrocellulose test-strip system as well as in solution.
• Expressed and purified beta-lactamase both with and without TALE-A (Bba_ K1189007) and (Bba_ K1189031) and characterized the functionality of these Biobricks as a reporter using both pH and ampicillin resistance assays.

Further Characterization and Improvement of Parts Already in the Registry

• Submitted optimized parts of TALE-A (Bba_ K1189022) and TALE-B (Bba_ K1189023) (Slovenia 2012) for use in E. coli by removing the eukaryotic Kozak sequence and nuclear localization signal, codon optimizing it for expression in E. coli and finally adding a KasI restriction site in the composite part such that future teams can essentially plasmid switch their own custom TALEs into our system. We also fixed a mutation in the binding region of TALE-B.
• Showed that the improved TALES were able to be expressed in E. coli.
• Submitted multiple composite parts of TALE-A and TALE-B with his-6 tags for easy purification, as well as with linkers and our two reporters: Beta-lactamase and ferritin.
• Expressed and successfully purified various TALE constructs: TALE-A (BBa_K1189000); TALE-B (BBa_K1189001); TALE-A with a K coil (BBa_K1189029); TALE-A linked to beta-lactamase (BBa_K1189031); TALE-A fused to ferritin (BBa_K1189021).
• Submitted the K coil (BBa_K1189010) and E coil (BBa_K1189011), previously in the registry, however lacking DNA. These coiled coils are very useful for in vitro assembly of different proteins and their ability to scaffold proteins together when fusion does not work, making an attractive addition to the registry. We also added a his-6 tag to these coils K coil with His-6 tag (Bba_K1189012) and E coil with a His-6 tag (Bba_K1189013). We submitted these coils in the Freiburg backbone for easy fusion of proteins.
• Characterized the binding of these coils, demonstrating that the coils allow in vitro assembly by doing a dot blot assay with our mobile TALE detector and Prussian blue reporter, both of which we have built, expressed, and purified with the coiled coils. These results can be found (here).
• Demonstrated that TALE-A with a K coil (BBa_K1189029) binds specifically to its target DNA. These results can be found (here).
• We were able to calculate the dissociation constant of TALE-A (BBa_K1189029) and TALE-B with coils in the FerriTALE complex.

Modelling

• Built an animation in Maya in order to communicate more visually how our sensor system functions from start to finish.
• Built a spatial model in Maya to show how the potential mechanism of DNA-TALE binding to nitrocellulose in our prototype could result in reduction of the TALEs sensitivity. This directly influenced the way we designed our system, encouraging us to add in linkers/scaffolds.
• Constructed a quantitative Mathematica model to test how amounts of target DNA versus detector proteins could influence sensitivity of the prototype.
• Built a Mathematica model to analyze kinetics of several common reporter enzymes found in the registry, which culminated in the selection of our reporters.
• Built a Scilab model of differential equations to show how fast we can get a detectable result from our system. This model used constants from our own experiments to make this prediction.

Human Practices

• Consulted with three key stakeholders in the beef-cattle industry and implemented their suggestions into our design in terms of where we would implement our test in the meat processing chain, the time requirements for our test as well as the need for ease of use and robustness in our final system
• Built upon previous outreach activities as well as took on a few new initiatives in order to engage more people in our community about our project

Collaboration

• Collaborated with Paris Bettencourt to create SensiGEM, a searchable database of biosensors. Here we identified all the different biosensors in the registry over the years and categorized them by their inputs and outputs in order to guide future teams in the selection of their biosensors.
• Helped establish the first Consort High School iGEM team in collaboration with the Calgary Entrepreneurial team. Consort performed amazingly well winning the Best Human Practices at the iGEM High School Jamboree!

Additional Work and Characterization

• Characterized a portable prototype showing that this final system is feasible. We used a homestyle pregnancy kit to show that this is possible here. We showed that it is possible to flow DNA through the strip, add protein on the strip and get a colour output using our ferritin reporter.