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 the beta-lactamase using ampicillin survival assays. We also characterized B-lac as a reporter by using phenol red as an indicator .

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 temperature and pH conditions. We also showed that Prussian blue ferritin is a good reporter by doing dot-blots with ferritin and horseradish peroxidase gives us an output that is quantifiable with the amount of ferritin that is present. 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 expressed, isolated and converted a TALE-ferritin fusion into TALE-Prussian-blue ferritin fusion.

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Further characterization of parts already present within the registry

In 2012 Wagenigen worked on and characterized E and K coils in their system. However, they did not submit the physical DNA for these parts. We thought these are useful parts that are great for scaffolding proteins together when fusion does not work. 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.

We showed that the coils bind to each other by doing a surrogate assay with a dot blot assay.

We 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.

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.