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Hamilton Path Problem with Golden Gate Shuffle

Hamilton Path Problem with Golden Gate Shuffle. Catherine Doyle, James Harden, Julia Fearrington , Duke DeLoache , Lilly Wilson. Solving the Hamilton Path Problem Through Golden Gate Shuffling. Golden Gate Shuffling vs. BioBricks. Golden Gate Shuffling. Bio Bricks. Pros

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Hamilton Path Problem with Golden Gate Shuffle

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  1. Hamilton Path ProblemwithGolden Gate Shuffle Catherine Doyle, James Harden, Julia Fearrington, Duke DeLoache, Lilly Wilson

  2. Solving the Hamilton Path Problem Through Golden Gate Shuffling

  3. Golden Gate Shuffling vs. BioBricks Golden Gate Shuffling Bio Bricks Pros Can flip single DNA fragment or multiple adjacent fragments in vivo Cons Scars Slow to build: days Can only add 1 edge at a time Attach each component through ligations Unintentional recombination with repeated hix sites • Pros • Allows us to split at any site with 4 bp in common • No scars or repeated hix sites • Make all the edges possible • Make all possible edge combinations • Feasible Selection processes • Quick 1-2 hrs • Cons • In vitro • More random assembly

  4. Build and Choose Edges 6 Edges 6 Half edges B2 Promoter RBS A1 B2 Promoter RBS A1 C2 Promoter RBS A1 C2 Promoter A2 B1 Promoter RBS C1 RBS C2 B1 Promoter RBS A2 Promoter B2 C1 Promoter RBS B1 RBS A2 C1 Promoter RBS

  5. Build and Screen for Solutions PCR w/ primers to create 6 half edges B2 Promoter RBS A1 choose subset of edges to build all possible orderings n times GGS B2 Promoter RBS A1 GGS N edges PCR Size Selection Clone into plasmid screen phenotypes

  6. Column Method (n+1 edges) PCR w/ primers to create n edges Each column must ligate in a particular position GGS z 4 4 4 X2 A1 4 4 4 B1 A2 A2 Y1 A2 B1 4 4 4 4 B1 C2 C2 B1 4 X2 B1 4 B2 Y1 4 4 4 4 B2 A1 B2 A1 4 C1 X2 4 4 4 4 Y1 C2 4 C2 A1 C2 A1 4 4 4 4 A2 C1 A2 C1 4 4 4 4 B2 C1 B2 C1 size of cloned insert pre-determined by number of columns GGS Clone and measure Phenotypes Solution(s) N Edges

  7. Alternative Column Method (n-1 edges) PCR w/ primers to create n edges Each column must ligate in a particular position GGS 4 4 4 4 B1 A2 A2 B1 4 4 4 4 B1 C2 C2 B1 plasmid contains promoter plus A1 plasmid contains k2 terminus 4 4 4 4 B2 A1 B2 A1 4 4 4 4 C2 A1 C2 A1 4 4 4 4 A2 C1 A2 C1 4 4 4 4 B2 C1 B2 C1 size of cloned insert pre-determined by number of columns GGS Clone and measure Phenotypes Solution(s) N Edges

  8. Conclusions • Could use GGS problems with Hin and Hixfor scar-less assembly • Start with first method and advance to the column method. • Using all 6 edges is not mathematically interesting but is biologically impressive • Will not use all 6 edges in experiment

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