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Team:Calgary/Project/DataPage
From 2013.igem.org
Data Page
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 < show here>.
We characterized ferritin as a reporter. Firstly, we converted ferritin into Prussian blue ferritin, which has high catalytic activity. In addition, this is a very attractive reporter because it is quite stable in a large range of temperature and pH conditions. Prussian blue ferritin uses conventional horseradish peroxidase substrates, ABTS and TMB. 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 good reporter 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 converted this ferritin into Prussian blue ferritin on which we have done intial characterization on nitrocellulose.
We have bulit and expressed beta lactamse in our bacterial cells which we have characterized using an ampicillin survival assays. We have also built the TALE A linked to beta lactmase, representing the alternative mobile unit in our sensor, which we have expressed and purified. This fusion protein has also been characterized with variations in the ampicillin assay. We have also done initial characterizations of the beta lactamse 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 made further improvement to TALE A ( Part:BBa_K782004) and TALE B ( Part:BBa_K782006) in our bigger constructs including J04500+His+TALEA+link+Kcoil ( BBa_K1189029), J04500+His+TALB+link+Kcoil ( BBa_K1189030), J04500+His+TALA+Link+Blac ( BBa_K1189031), and J04500+His+TALA+hFTN+lFTN ( BBa_K1189021):
Slovenia 2012 iGEM team did a great characterization of TALE A ( Part:BBa_K782004) and TALE B ( Part:BBa_K782006). We used these parts as a proof of concept of our system. Along the way we made some changes to these parts to make them more useful to us and the parts registry. Slovenia 2012 iGEM team used these parts in eukaryotic cells. Therefore, these parts had a kozak sequence before the start of the gene.
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 did not submit the physical DNA for the coils. 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 submitted these parts K coil(Bba_K1189027) and E coil (Bba_K1189011). We also added these parts with a his-6 tag such that fusion proteins with K or E coil can be purified out using affinity chromatography. These four parts have been submitted in the Freiburg fusion biobrick backbone to allow easier construction of fusion protein for future teams. 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.
We have also mutated, optimized, and repurposed Slovenia’s TALE proteins from 2012 to work in E. coli . The TALE that Slovenia submitted had mutations, as it is difficult to sequence RVDs in TALEs. We amended the TALE to fit the correct binding sequence. This was not optimized to work in E. coli, it had a eukaryotic ribosomal binding site called a Kozak sequence. In the presence ok Kozak sequence the TALEs are not expressed in E. coli . We removed the Kozak sequence and codon optimized the TALEs for expression in E. coli. Further, we characterized the TALEs and showed that they bind to the correct nucleotide sequences.
Something about the kas1 site that ALi put in
Modelleing
Quantitative and qualitative
Collaboration
Sensigem
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.
