1. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 1 Jiyoun Lee

2. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 2 Introduction TSP (Traveling Salesman Problem) –Graph with given vertexes, edges, and costs (weights)  Hamiltonian paths containing the smallest sum of costs Airlines, transportation Graph dimension –7 vertexes, 24 edges, 5 costs

3. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 3 1 0 3 2 5 6 4 0  1  2  3  4  5  6  0 (21) start 3 5 3 3 7 11 3 3 9 3 3 end 0  1  6  5  4  3  2  0 (31) 0  2  1  3  4  5  6  0 (27) 0  2  3  4  5  6  1  0 (31) 0  6  5  4  3  1  2  0 (27) 0  6  5  4  3  2  1  0 (21)

4. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 4 vertexweight (with low GC) vertexweight (with low GC) vertex edge low melting temperature weight vertexweightvertexweightvertexweight low melting temperature more amplification by more denaturation

5. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 5 ds DNA denaturation : more denaturation of DNA strands which have less weights primer annealing polymerization

6. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 6 Procedure Generating random paths through the graph Keep only those paths that begin with v in Keep only those paths that enter exactly n vertices Keep only those paths that enter all of the vertices of the graph at least once Separation of the solutions Generating sequences & LIgation reaction PCR with v in as primers Gel electrophoresis separation by size Affinity-purification with a biotin -avidin magnetic beads system TGGE Figure out the pathSequencing & readout

7. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 7 Procedure Generating random paths through the graph Keep only those paths that begin with v in Keep only those paths that enter exactly n vertices Keep only those paths that enter all of the vertices of the graph at least once Separation of the solutions Generating sequences & LIgation reaction PCR with v in as primers Gel electrophoresis separation by size Affinity-purification with a biotin -avidin magnetic beads system TGGE Figure out the pathSequencing & readout

8. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 8 Affinity separation (7 nodes) Ligation (Connecting each ssDNA) PCR with Vin Hybridization (Generating all possible paths ) Size separation by electrophoresis Sequencing Temperature gradient in reservoir Isothermal reaction in reservoir Expected problems : buffer composition Ligation buffer Affinity separation buffer Cf) conventional method  Purification of DNA by ethanol precipitation Paramagnetic separation using biotin-avidin interaction Temperature cycling in reservoir CE (or CGE)

9. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 9 Affinity unit Reaction chamber for hybridization and ligation Reaction chamber for PCR Electrophoresis separation Buffer change Volume reduction? I wish to.. Eliminate the affinity step Change the buffers in flow

10. Cell and Microbial Engineering Laboratory Solving TSP on Lab-on-a-Chip 10 Questions and Discussions… Danny’s idea: addition of salts.. –Not reservoir type, but channel type In English, please… Questions to Danny: –Method for inserting beads to channel. –Channeling effect or something… –TRIS,.. Some buffer names.. –Many beads – Yield of …