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.

Bioinformatics

55,000 Potential 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

Sequencing results of Library transformation

Error rates

• Perfect sequence matches a designed ZF: 57.1% (44/77)
• Single SNP: 2.6% (2/77)
• Two or more SNPs: 18.2% (14/77)
• Frame shift: 22.1% (17/77)

We determined the overall per base pair error rate for this set sequenced to be around 1/200, which includes errors generated by the chip, or generated during PCR and assembly. These are a bit higher than those found by Kosuri, et al., but within a reasonable margin.

Distributions

Of the 77 samples with good sequencing results, 2 sequences were repeated once. Discounting these, 73 of the 77 sequences, or 94.8%, were unique, showing substantial variability within the library.