Post-Regionals

Sensor: Putting it all together

Can we detect DNA with specificity?

Can we successfully capture our DNA with our detectors with specificity and report it?

This assay shows that we can capture our target DNA with two detector TALEs with specificity . Additionally, we can report whether that DNA has been captured and is present in the sample, which is a very important concept for our sensor system.

Can our coils bind?

After putting in gratuitous effort to build parts containing these coils and successfully purifying these proteins we wanted to determine if the E and K coils interacted with each other. In order to characterize coil-coil interaction we performed an immunoprecipitation (IP) assay. We built a GFP with an E-coil ( BBa_K1189014) and we also built TALE-B with a K-coil (BBa_K1189030). To characterize the binding of the coils we pulled down with either an immunoglobulin G antibody (IgG) that serves as a negative control or with GFP antibody. The idea behind this experiment is to pull down the E coil which is fused to GFP with a GFP antibody and then probe with a anti-his antibody which recognizes the his tag on the TALE fused to the K coil. Upon interaction between the E and K coils we will see an output at approximately 86 kDa when probed with a His-antibody as seen in Figure 7. Our test groups included the coils by themselves and both the E and K coils put together in solution. As seen in Figure 7 a band appears only when we pull E and K coil with a GFP and probe with an anti-his antibody indicating the presence of both GFP and TALE in the elution solution indicating that the coils interact with each other.

How does the use of Coils versus Direct Fusions of TALEs Affect our Prussian Blue reporter?

After successfully confirming that we could convert our own ferritin proteins that were produced from the parts we constructed (BBa_K1189018, BBa_K1189021) into Prussian blue ferritin the next step was to evaluate how the design of our parts could potentially affect the reporter activity of our Prussian blue ferritin. Based on the spatial modelling performed by our team it was suggested that assembly of the ferritin nanoparticle with TALE proteins directly fused was highly unlikely. This is because the TALE proteins are significantly larger than the ferritin subunits. Their size would likely result in steric hindrance and prevent the assembly of the full ferritin protein. In order to test the predictions put forward by our modelling we ensured that our protein samples were balanced in order to have the same number ferritin cores in each sample. The catalytic activity of these proteins was then compared. From the data gathered we saw that the Prussian blue ferritin with fused coils (even if TALES are additionally bound to the ferritin via coils) was more effective as a reporter than having the TALE proteins directly fused to the ferritin nanoparticle (Figure 19). The results from this experiment suggest that the predictions made by our model were correct. Using coils however alleviates this issue as these coils are small and would not interfere in the ferritin self-assembly but can be used to attach our TALES to create the FerriTALE.

Further Characterization of our parts

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