Data Page

Characterization of new parts submitted to the Registry

• We submitted two new reporters to the registry, Beta-lactamase (Bba_ K118908) and Recombinant Human Ferritin. Ferritin was submitted in its individual subunits, (Bba_ K1189024), its individual subunits (Bba_ K1189025), as well the two fused together We submitted these parts alone, as well as with 6-his tags: (Bba_ K118909) and (Bba_ K118908)
• We expressed (Bba_ K118918), which possess 24 subunits, and showed successful chemical conversion of the expressed protein into Prussian blue ferritin.
• We characterized (Bba_ K118918) as a reporter using two different substrates and a variety of different conditions. W
• We also showed that (Bba_ K118918) is a feasible reporter for our system by doing dot-blots on nitrocellulose and our model prototype (see below) to give us both quantitative and qualitative outputs.
• We have cloned, expressed and purified beta lactamase (Bba_ K118908) and characterized its fuctionality as a reporter using multiple assays.
• We constructed fusions of TALE-A and TALE-B, previously submitted by the TALE A linked to beta lactamase, representing the alternative mobile unit in our sensor, which we have expressed and purified. We characterized this fusion protein with an ampicillin assay . We have also done initial characterizations of the beta lactamase as a reporter by using benzylpenicillin as a substrate which gives a pH output. When combined with phenol red as a pH indicator, we have shown a colourimetric output which correlates to changing amounts of beta lactamase.
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Further characterization and improvement of parts already present within the registry

• We optimized the expression of TALEs (TALE-A and TALE-B) that Slovenia 2012 used 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 TALEs into our system. We also fixed a mutation in TALE-B.
• We cloned, expressed and purified composite parts with these TALEs and submitted them with an IPTG inducible promoter and a his-6 tag for easy purification ( BBa_K1189000, BBa_K1189001).
• Slovenia 2012 iGEM team conducted all their assays in vivo. We showed that TALE-A and TALE-B can be expressed in E. coli, and purified for use in vitro here.
• We expressed and successfully purified the following parts: TALE-A ( BBa_K1189000); TALB ( BBa_K1189001); TALE-A with a K-coil ( BBa_K1189029); TAL-A linked to beta-lactamase ( BBa_K1189031); TAL-A fused to ferritin (BBa_K1189021).

• In 2012, the Wageningen team worked with and did initial characterized a coiled coil system known as the E and K coils for their project. However, they were unable to submit the physical DNA for the coils. We submitted the K-coil ( BBa_K1189010) and E-coil( BBa_K1189011). 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 Freidberg backbone for easy fusion of proteins.
• We have done initial characterization 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 here but FIX THIS LINK.

Modelleing

• 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.

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.

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 LINK 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.