We did not have time to test all 6 targets as planned: we chose to focus on zinc finger binders to DNA tripplet TGG, which is found in the color blindness gene bottom target.

Color Blindness (CB) Bottom Hits

We assembled the Color Blindness Bottom library, transformed it into the proper selection strain, and plated on incomplete media with varying concentrations of 3-AT. We saw colonies form, with more on the less stringent plates (NM) and fewer colonies on the higher 3-AT concentrations. Several colonies were picked and tested under varying concentrations of 3-AT in plate reader overnight, and there was a clear difference in growth between the colonies and the negative control (CB bottom binding site with Zif268).

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These colonies were then sequenced to discover which of our library zinc fingers was successfully binding to the 5'-GTG GGA TGG-3' sequence. So far, several different novel zinc fingers have been identified! All of the sequenced colonies used the Zif268 backbone, but since the TGG codon is not unlike known Zif268-based zinc finger binding sequences, this may imply that Zif268 is the optimal backbone for such base triplet. The actual DNA-binding helix is much more variable, and we will continue to test the strength of the binding interactions of these zinc fingers and look for trends in their amino acid make-up.