Part:BBa_K1189014:Experience

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Improvements

Team Wageningen UR 2012 worked with E/K coil pairs, however only submitted a single E coil fused to GFP. As these parts are only useful when the complementary coil is used, and order for these parts to be more capable of being used for a wide variety of dimerization and tagging purposes on any protein of interest, we have submitted the E and K coil parts on their own (with Frieburg cutsites to allow for protein fusions to be created), with His tags in order to allow for protein purification, and with GFP fusions to allow for tagging of proteins with a reporter.

Applications of BBa_K1189010

We evaluated the binding of our coils using other constructs that make use of the E and K coil parts submitted. In the case of the coils we were interested to see if the K-coil fused to TALE proteins (BBa_K1189029, BBa_K1189030) could bind to the E-coil found on one of our Prussian blue ferritin constructs (BBa_K1189018). To complete this task we placed the TALE on the membrane, washed and blocked the membrane. The ferritin protein with the complimentary coil was then added to the membrane. If this coil successfully binds to the other coil then the ferritin will not be washed off during the next wash step. We can then see if Prussian blue ferritin is bound by adding a TMB substrate solution that will cause a colour change. To this extent we saw a blue ring in this trial indicating a positive result. This suggests that our coils are actually binding in an in vitro system.

Another interesting element of this assay is why we used two variants of the TALE K-coil negative control. A blue ring on our TALE negative control confirmed our fear that during the second protein application and wash step that some of the ferritin with coil proteins would drift over and bind to the TALE K-coils on the nitrocellulose. This did not occur for our separate negative control (Figure 3).

Figure 3. This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately. Image contrast was altered to make the results more clear on a digital monitor; the same changes were applied to each element of the figure.

We also performed an immunoprecipitation assay to demonstrate the binding of the E/K coils (Figure 4).

Figure 4. Assay showing coiled-coil interaction in vitro. Crude lysates from a negative control (RFP), GFP-Ecoil and His-Kcoil were combined together to investigate interaction and immunoprecipitated with GFP or an isotype control and then further probed with α-His antibody. Only in the presence of both GFP and a His tag we see a band indicating interaction.

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