Data Page

Characterization of new parts submitted to the Registry

• This year we submitted two new reporters to the registry, Beta-lactamase (Bba_ K118908) and Recombinant Human Ferritin (Bba_ K118918). We built the parts, expressed, and purified these proteins < here>.
• Firstly, we converted ferritin into Prussian blue ferritin, which has high catalytic activity and then characterized it as a reporter. We tested Prussian Blue ferritin using two different substrates, ABTS and TMB, a range of temperatures and pH conditions. We did a Michelis-Menten kinetic analysis with Prussian blue ferritin and characterized it under ranging temperature and pH conditions.
• We also showed that Prussian blue ferritin is a feasible reporter in our system by doing dot-blots on nitrocellulose and our model prototype (see below) with its substrates to give us a quantitative and qualitative output, respectively. In addition, we showed that ferritin, a protein with 24 subunits is expressed and functional in our system.
• We have also expressed, isolated and converted human recombinant ferritin into Prussian blue ferritin on which we have done initial characterization on nitrocellulose.
• We have cloned, expressed and purified beta lactamase in our bacterial cells which we have characterized using an ampicillin survival assays.
• We have also built 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.
.

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_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_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 Retrieved from "http://2013.igem.org/Team:Calgary/Project/DataPage"