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Published byFrancesca Eastman Modified about 1 year ago

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Hamilton Path Problem with Golden Gate Shuffle Catherine Doyle, James Harden, Julia Fearrington, Duke DeLoache, Lilly Wilson

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Solving the Hamilton Path Problem Through Golden Gate Shuffling

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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

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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

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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

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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 B1A2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Y1A2 4 4 Y1 C2 4 4 Y1B2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 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

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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 B1A2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 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

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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

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