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>.
• 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.
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Further characterization and improvement of parts already present within the registry

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.

Human practices