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Transcript
Several coupled DNA and protein libraries are constructed, randomizing 3 base pairs and 5 contacting amino acids for each NNNGGAGGTTTCTCTGTAAA TGANNNGGTTTCTCTGTAAA TGAGGAGGTTTCTCTGTNNN Library-on-library cleavage profiling Double-stranded target pool is used to stain yeast Cleavable targets on cleaving enzymes are cut, rest remain intact Biotinylated linker is ligated to cleaved targets HE-expressing yeast Streptavidin purification removes yeast and non-cleaved targets Recovering sequences PCR amplify ligated, purified oligos TA clone Colony sequencing will result in a full target profile! Can then extract plasmids and create dual-conjugated target DNA for any sequence recovered Matching proteins to targets Create yeast library, oligo pool + + Recover cleavable sequences Choose desired sequence, PCR amplify (with conjugates for FACS) Now haveclone(s) Recover which cleave this matched sequence protein/target pair
