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 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 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

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

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

Column Method (n+1 edges) z z A1 X2 4 4 C1 X2 4 4 B1 X2 4 4 B1 A2 4 4 A1B2 4 4 B1C2 4 4 C1B2 4 4 C1A2 4 4 A1C2 4 4 A1B2 4 4 B1 C2 4 4 C1B2 4 4 C1A2 4 4 A1C2 4 4 B1A Y1A2 4 4 Y1 C2 4 4 Y1B GGS N Edges Clone and measure Phenotypes Solution(s) GGS PCR w/ primers to create n edges Each column must ligate in a particular position PCR w/ primers to create n edges Each column must ligate in a particular position size of cloned insert pre-determined by number of columns

Alternative Column Method (n-1 edges) B1 A2 4 4 A1B2 4 4 B1C2 4 4 C1B2 4 4 C1A2 4 4 A1C2 4 4 A1B2 4 4 B1 C2 4 4 C1B2 4 4 C1A2 4 4 A1C2 4 4 B1A GGS N Edges Clone and measure Phenotypes Solution(s) PCR w/ primers to create n edges Each column must ligate in a particular position PCR w/ primers to create n edges Each column must ligate in a particular position GGS plasmid contains promoter plus A1 plasmid contains k2 terminus size of cloned insert pre-determined by number of columns

Conclusions Could use GGS problems with Hin and Hix for 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