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 (Bba_ K118918).
• 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 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.
.

Further characterization and improvement of parts already present within the registry

• We optimized the expression of TALEs 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 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 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 Wagenigen 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 Friedburg 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

CHRIS WILL PUT THIS UP

Human Practises

• Consulted with three key stakeholders in the beef-cattle industry and catered our system as an addition to the already existing testing for enterohemmorhagic E. coli.
• Collaborated with Paris Bettencourt to create sensiGEM where we identified all the different biosensors in the registry over the years and categorized them by their inputs and outputs. We hope this will help guide future teams in the selection of their biosensors.

Additional Work and Characterization

We 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 ferritin. ATTN: ROBERT