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Team:Harvard/Results
From 2011.igem.org
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==Genome-Based Selection System== | ==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. | 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. | ||
| + | [[File: HARVhybrid2_cropped.png|300px|right]] | ||
=Zinc Finger Binders= | =Zinc Finger Binders= | ||
Revision as of 20:33, 25 September 2011
Overview | MAGE | Lambda Red| Chip-Based Library | One-Hybrid Selection | Zinc Finger Binders | Biobricks
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:
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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.
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 xx novel zinc fingers that successfully bind to 5'-GTGGGATGG-3'.
