Latest revision as of 07:43, 2 November 2013

TALE B fused to C-terminus K coil with his tag

For information on TALE B refer to BBa_K1189023.

E/K coils are synthetic coiled-coil domains designed specifically to bind to each other with high affinity and specificity (Litowski and Hodges, 2002) (Figure 1). They are composed of a heptad repeat that forms a coil structures that are able to interact with each other. These coils are able to interact with each other in an anti-parallel fashion that makes them useful for applications such as peptide capture, protein purification and in biosensors. For our project we decided to make use of the IAAL E3/K3 coils (BBa_K118901, BBa_K1189011) due to the balance they offer between affinity and specificity (Table 1).

These E3/K3 coils are able to form heterodimers due to the hydrophobic residues contained within the heptad repeat. In our case these are isoleucine and leucine residues. Designated by empty arrows in the helical wheel diagram below (Figure 2) these residues form the core of the binding domain of the coils. In order to prevent the homodimerization of these coils charged residues are included in the design. The electrostatic interactions between glutamic acid and lysine residues prevent an E-coil from binding with an E-coil for example. These parts were already in the registry, however the DNA was never received, so we built, sequenced and re-submitted them.

Results

Expression from pSB1C3

The 2013 iGEM Calgary successfully expressed and purified this protein in pSB1C3 and per this part sequence exactly using and FPLC and metal affinity purification of the his tag. See Figure 4 for an SDS-PAGE of this 88 kDa isolate.

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).

We also performed an immunoprecipitation assay to demonstrate the binding of the E/K coils (Figure 6).

TALE B sequence binding and kinetics

We ordered 60mer FAM-labeled [B] (TALE B target sequence) oligos and hybridized them with their reverse complement oligo to make double stranded pieces of DNA containing the target sequence of our TALEs. Using these target sequences and following the TALE Nitrocellulose Functionality Assay, we showed that TALEB binds to its target sequence. We incubated Ferritin fused to an Ecoil (BBa_K1189018) to TALE fused to a Kcoil (BBa_K1189030) to make the FerriTALE complex. The complex was then blotted on strips of nitrocellulose paper. The strips were then blocked with milk and soaked in the appropriate DNA solution. Finally, the strips were washed and imaged (figure 5). We performed a densitometery test on these results and were able to calculate the dissociation constant of the TALEs.

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