Novel Zinc Fingers

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. The TGG library was assembled and transformed into the selection strain with the proper binding site. So far we have screened 26 colonies and found 15 possible novel zinc finger binders to the sequence 5'-GTGGGATGG-3'. See here for more details.

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One-Hybrid Selection Strain

Genome-Based Selection System

We designed a one-hybrid metabolic system that was entirely genome-based. Using multiplex automated genome engineering (MAGE) and lambda red, we knocked out HisB, PyrF, and rpoZ; inserted a kanamycin cassette-zinc finger binding site-His3-URA3 construct into the 1529620 locus; and changed the zinc finger binding site directly on the genome. The strain was fully characterized and was sensitive enough to recognize a valid zinc finger when diluted as much as one into one million of negative controls. See here for more details.