Zinc Finger Binders

The Color Blindness Bottom library was assembled and transformed into the selection strain with the proper binding site. So far we have screened 26 colonies and found 18 possible novel zinc fingers that successfully bind to 5'-GTGGGATGG-3'.

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.

Bioinformatics

55,000 Possible Zinc Fingers

We made 55,000 sequences, distributed evenly among 6 DNA target triplets. That's 9150 per target.

Because our program's output changes dramatically based on the input triplet, no two sets of sequences are the same:

AAA	ACC	CTC
CTG	GAC	TGG