1. E.HOP: A Bacterial Computer to Solve the Pancake Problem Davidson College iGEM Team Lance Harden, Sabriya Rosemond, Samantha Simpson, Erin Zwack Faculty advisors: Malcolm Campbell, Karmella Haynes, Laurie Heyer

2. Basic Pancake Parts NameIconDescription J31009pSB1A7 (insulated plasmid) J31000Hin Recombinase J31001Hin Recombinase-LVA J31011Recombination Enhancer J44000HixC J44001RBS reverse J31003Kan R forward J31002Kan R reverse J31005Chl R forward J31004Chl R reverse J31007Tet R forward J31006Tet R reverse J44002pBAD reverse J31011RFP and RBS reverse RBS Tet Kan Chl Davidson Missouri Western HinLVA Hin Kan Chl Tet

3. Basic Pancake Parts AvailableTestedIncluded in Devices J31009pSB1A7 (insulated plasmid) J31000Hin Recombinase J31001Hin Recombinase-LVA J31011Recombination Enhancer J44000HixC J44001RBS reverse J31003Kan R forward J31002Kan R reverse J31005Chl R forward J31004Chl R reverse J31007Tet R forward J31006Tet R reverse J44002pBAD reverse J31011RFP and RBS reverse RBS Tet Kan Chl Davidson Missouri Western HinLVA Hin Kan Chl Tet

4. Define a Biological Pancake Nonfunctional Functional RBS pBad Tet hixC RE hixC T T T T pancake 1pancake 2 pBad Tet hixC RE hixC T T T T pancake 1pancake 2 RBS

5. Modeling Pancake Flipping 5 C 2 = 10 ways to choose two spatula positions Assume all 10 ways are equally likely Simulate random flipping process with MATLAB -2 4 3

6. Modeling 8 Stacks of 2 Pancakes ( 1 2) (-1 2) ( 1 -2) (-1 -2) ( 2 1) (-2 1) ( 2 -1) (-2 -1) *Solution is (1, 2) or (-2, -1)

7. ( 1, 2) (-2, -1) ( 1, -2) (-1, 2) (-2, 1) ( 2, -1) (-1, -2) ( 2, 1) Modeling 8 Stacks of 2 Pancakes *Solution is (1, 2) or (-2, -1)

8. Modeling 8 Stacks of 2 Pancakes ( 1, 2) (-2, -1) ( 1, -2) (-1, 2) (-2, 1) ( 2, -1) (-1, -2) ( 2, 1) *Solution is (1, 2) or (-2, -1)

9. Four One-Pancake Constructs Predicted Tet Resistance 1 (2) 1 (-2) (1) 2 (-1) 2 + Tet T T T T RBS Tet T T T T RBS Tet T T T T RBS T T T T - - + Observed Tet Resistance + + + + Tet RBS

10. How to Detect Flipping via Restriction Digest NheI 200 bp NheI 1 (2) 1 (-2) (1) 2 (-1) 2 Tet T T T T RBS Tet T T T T RBS Tet T T T T RBS T T T T Tet RBS 300 bp 1100 bp Expected Fragment Size

11. Tet Pancake Flipping pBad Pancake Flipping Hin-mediated Flipping (1) 2 (-1) 2 (1) 2 + Hin 1 (2) 1 (-2) 1 (2) + Hin

12. Read-Through Transcription Blocked by pSB1A7 Observed Tet Resistance in pSB1A3 + (1) 2 + (-1) 2 + 1 (2) + 1(-2) ampR rep(pMB1) T T T T T T T T device pSB1A7 EX SP T T

13. ampR rep(pMB1) T T T T T T T T device pSB1A7 EX SP T T Observed Tet Resistance in pSB1A7 + (1) 2 - (-1) 2 + 1 (2) - 1(-2) Read-Through Transcription Blocked by pSB1A7 Observed Tet Resistance in pSB1A3 + (1) 2 + (-1) 2 + 1 (2) + 1(-2)

14. Original Design Problems RBS Tet hixC T T T T pBadREpLac Hin LVA RBS T T T T AraC PC

15. Flips too fast Original Design Problems RBS Tet hixC T T T T pBadREpLac Hin LVA RBS T T T T AraC PC

16. Unclonable Original Design Problems RBS Tet hixC T T T T pBadREpLac Hin LVA RBS T T T T AraC PC

17. #1: Two pancakes (Amp vector) #2: AraC/Hin generator (Kan vector) AraC PC T T T T T T T T pLac RBS Hin LVA pSB1K3 hixC RBS hixC Tet hixCpBad pSB1A7 Two-Plasmid Solution

18. RBS Tet RBS Tet LB Amp + Kan+ Tet (1,2) (-2,-1) Biological Equivalence Problem

19. RBS Tet (1,2) (-2,-1) LB Amp + Kan+ Tet Biological Equivalence Solution RBS Tet (1,2) (-2,-1) RFP RBS RFP RBS

20. (1,2) (1,-2) (-1,2) (-1,-2)(2,1) (2,-1) (-2,-1) Eight Two-Pancake Stacks (-2,1)

21. Intermediate Results Amp, Kan, Tet + AraC/ Hin Tet Resistance Hin Vector + IPTG Hin Induction Amp + Tet Tet Resistance (1, 2)  Hin-LVA + (1, 2) + (1,-2) (-1,-2)  AraC/Hin-LVA + (1,-2) (-1,-2)  Screen for Orientation Repress pBad-Tet Activate pLac-Hin

22. PRACTICAL Proof-of-concept for bacterial computers Data storage n units gives 2 n (n!) combinations BASIC BIOLOGY RESEARCH Improved transgenes in vivo Evolutionary insights CONCLUSIONS: Consequences of DNA Flipping Devices -1,2 -2,-1 in 2 flips! gene regulator

23. Better control of kinetics Slow down Hin activity Determine x flips second -1 Size bias? Next Steps Number of flips vs.Time? Number of flips Time

24. Modeling Plasmid Copy Number Copy ## Solutions Required P(plasmid solved) P(cell survives) 20010.251 - (1 - 0.25) 200 ≈ 1 20010.0031 - (1 - 0.003) 200 ≈ 0.45 20010.011 - (1 - 0.01) 200 ≈ 0.87 P(cell survives) = n = plasmid copy number p = probability of a pancake stack being sorted x = number of sorted stacks sufficient for cell survival

25. Improved insulating vector Accepts B0015 double terminator Bigger pancake stacks Next Steps

26. Multiple campuses increase capacity with parallel processing Collective Troubleshooting Primarily Undergraduate Institutions can iGEM collaboratively Summary: What We Learned

27. Math and Biology mesh really well Math modeling We proved a new theorem! Challenges of biological components Summary: What We Learned

28. But above all… We had a flippin’ good time!!!

29. E.HOP: A Bacterial Computer to Solve the Pancake Problem Collaborators at MWSU: Support: Davidson College HHMI NSF Genome Consortium for Active Teaching James G. Martin Genomics Program Marian Broderick, Adam Brown, Trevor Butner, Lane Heard, Eric Jessen, Kelly Malloy, Brad Ogden

30. Extra Slides >>>

31. Modeling 384 Stacks of 4 Pancakes 2 n (n!) = X Combinations 2 4 (4!) = 384 Combinations

32. * * * * * * Build one representative of each family Modeling 384 Stacks of 4 Pancakes

33. Modeling Random Flipping for trial = 1 to n trials stack = input_stack flips = 0 while stack ~= 1:k flips = flips + 1 int = choose_random_interval stack = [stack(1:(int(1)-1)) -1 * fliplr(stack(int(1):int(2))) stack(int(2)+1:k)] end

34. 1 2 -1 2 1 -2 -1 -2 -2 1 -2 -1 2 -1 2 1 P 2 : 2 Burnt Pancakes 8 vertices, each with degree 3 Flip length 1 Flip length 2

35. 1 2 -1 2 1 -2 -1 -2 -2 1 -2 -1 2 -1 2 1 P 2 : 2 Burnt Pancakes 8 vertices, each with degree 3 A 1212 -1 2 -1 -2 1 -2 -2 -1 -2 1 2121 2 -1 1 2 01011000 -1 2 10100100 -1 -2 01010010 1 -2 10100001 -2 -1 10000101 -2 1 01001010 2 1 00100101 2 -1 00011010

36. 1 2 -1 2 1 -2 -1 -2 -2 1 -2 -1 2 -1 2 1 B 2 : 2 Burnt Pancakes 8 vertices, each with degree 3 A2A2 1212 -1 2 -1 -2 1 -2 -2 -1 -2 1 2121 2 -1 1 2 30200202 -1 2 03022020 -1 -2 20300202 1 -2 02032020 -2 -1 02023020 -2 1 20200302 2 1 02022030 2 -1 20200203