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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
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Basic Pancake Parts Name Icon Description Davidson Missouri Western
J31009 pSB1A7 (insulated plasmid) J31000 Hin Recombinase J31001 Hin Recombinase-LVA J31011 Recombination Enhancer J44000 HixC J44001 RBS reverse J31003 KanR forward J31002 KanR reverse J31005 ChlR forward J31004 ChlR reverse J31007 TetR forward J31006 TetR reverse J44002 pBAD reverse RFP and RBS reverse Hin Davidson Missouri Western HinLVA RBS Kan Kan Chl Chl Tet Tet
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Basic Pancake Parts Available Tested Included in Devices Davidson
J31009 pSB1A7 (insulated plasmid) J31000 Hin Recombinase J31001 Hin Recombinase-LVA J31011 Recombination Enhancer J44000 HixC J44001 RBS reverse J31003 KanR forward J31002 KanR reverse J31005 ChlR forward J31004 ChlR reverse J31007 TetR forward J31006 TetR reverse J44002 pBAD reverse RFP and RBS reverse Hin Davidson Missouri Western HinLVA RBS Kan Kan Chl Chl Tet Tet
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Define a Biological Pancake
Nonfunctional Tet RBS T hixC pBad hixC hixC RE pancake 1 pancake 2 Tet Functional RBS T hixC pBad hixC hixC RE pancake 1 pancake 2
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Modeling Pancake Flipping
-2 4 -1 3 5C2 = 10 ways to choose two spatula positions Assume all 10 ways are equally likely Simulate random flipping process with MATLAB
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Modeling 8 Stacks of 2 Pancakes
*Solution is (1, 2) or (-2, -1) ( 1 2) (-1 2) ( 1 -2) (-1 -2) ( 2 1) (-2 1) ( 2 -1) (-2 -1)
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Modeling 8 Stacks of 2 Pancakes
*Solution is (1, 2) or (-2, -1) ( 1, 2) (-2, -1) ( 1, -2) (-1, 2) (-2, 1) ( 2, -1) (-1, -2) ( 2, 1)
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Modeling 8 Stacks of 2 Pancakes
*Solution is (1, 2) or (-2, -1) ( 1, 2) (-2, -1) ( 1, -2) (-1, 2) (-2, 1) ( 2, -1) (-1, -2) ( 2, 1)
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Four One-Pancake Constructs
Predicted Tet Resistance Observed Tet Resistance + (1) 2 Tet T RBS + (-1) 2 Tet T RBS - 1 (2) Tet T RBS + 1 (-2) T Tet RBS -
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How to Detect Flipping via Restriction Digest
NheI NheI Expected Fragment Size (1) 2 Tet T RBS 200 bp (-1) 2 Tet T RBS 300 bp 1 (2) Tet T RBS 200 bp 1 (-2) T Tet RBS 1100 bp
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Hin-mediated Flipping
pBad Pancake Flipping Tet Pancake Flipping (1) 2 + Hin 1 (2) + Hin (1) 2 (-1) 2 1 (2) 1 (-2)
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Read-Through Transcription Blocked by pSB1A7
Observed Tet Resistance in pSB1A3 + (1) 2 + (-1) 2 + 1 (2) + 1(-2) ampR rep(pMB1) T device pSB1A7 EX SP
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Read-Through Transcription Blocked by pSB1A7
Observed Tet Resistance in pSB1A3 + (1) 2 + (-1) 2 + 1 (2) + 1(-2) ampR rep(pMB1) T device pSB1A7 EX SP Observed Tet Resistance in pSB1A7 + (1) 2 - (-1) 2 + 1 (2) - 1(-2)
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Original Design Problems
RBS Tet hixC T pBad RE pLac Hin LVA AraC PC
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Original Design Problems
pSB1A7 Unclonable RBS Tet hixC T pBad RE pLac Hin LVA AraC PC
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Original Design Problems
RBS Tet hixC T pBad RE pLac Hin LVA AraC PC Flips too fast
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Two-Plasmid Solution #1: Two pancakes (Amp vector)
#2: AraC/Hin generator (Kan vector) Tet RBS pSB1A7 hixC pBad hixC hixC AraC PC Hin LVA T RBS T pSB1K3 pLac
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Biological Equivalence Problem
RBS Tet (1,2) LB Amp + Kan+ Tet RBS Tet (-2,-1)
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Biological Equivalence Solution
RFP RBS RBS Tet (1,2) (1,2) LB Amp + Kan+ Tet RFP RBS RBS Tet (-2,-1) (-2,-1)
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Eight Two-Pancake Stacks
(1,2) (-2,-1) (2,-1) (1,-2) (2,1) (-2,1) (-1,2) (-1,-2)
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Screen for Orientation
Intermediate Results Amp, Kan, Tet + AraC/ Hin Tet Resistance Hin Vector + IPTG Hin Induction Amp + Tet (1, 2) Hin-LVA + (1, 2) + (1,-2) (-1,-2) AraC/Hin-LVA + Repress pBad-Tet Activate pLac-Hin Screen for Orientation
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CONCLUSIONS: Consequences of DNA Flipping Devices
-1, ,-1 in 2 flips! PRACTICAL Proof-of-concept for bacterial computers Data storage n units gives 2n(n!) combinations BASIC BIOLOGY RESEARCH Improved transgenes in vivo Evolutionary insights Possibly the first in vivo controlled inversion of DNA Act as a switch to turn a gene on and off Help determine the function and necessity of a gene Possible application for the rearrangement of transgenes in vivo Help to give insight about the mechanism of gene rearrangement Evolutionary insight Possible replacement of knockout mice Proof of concept for bacterial computers Power of having millions of parallel processors gene regulator
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Next Steps Better control of kinetics Slow down Hin activity
Determine x flips second-1 Size bias? Number of flips vs.Time? Number of flips Time Controlling kinetics Control the rate of inversion Allow for manipulation of the system Control the expression of the gene based on the environment Provide information about intermediate configurations of the inverted DNA before the final configuration is reached
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Modeling Plasmid Copy Number
P(cell survives) = n = plasmid copy number p = probability of a pancake stack being sorted x = number of sorted stacks sufficient for cell survival Number of Pancakes Number of Plasmids Number Solutions Required P (solution) P (cell survives) 2-stack 200 1 0.25 1 - ( )200 ≈ 1 4-stack 0.003 1 - ( )200 ≈ 0.45 different 4-stack 0.01 1 - ( )200 ≈ 0.87
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Next Steps Improved insulating vector Bigger pancake stacks
Accepts B0015 double terminator Bigger pancake stacks
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Summary: What We Learned
Multiple campuses increase capacity with parallel processing Collective Troubleshooting Primarily Undergraduate Institutions can iGEM collaboratively Troubleshooting “Insanity: doing the same thing over and over again and expecting different results.”- Albert Einstein Redesigning and execution Teamwork Division of labor amongst campuses Communication and Cooperation Intercampus Allows for optimal efficiency in the lab Intracampus Division of labor effectively Increased the amount of new ideas Increased productivity Size of the school is not important
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Summary: What We Learned
Math and Biology mesh really well Math modeling We proved a new theorem! Challenges of biological components Troubleshooting “Insanity: doing the same thing over and over again and expecting different results.”- Albert Einstein Redesigning and execution Teamwork Division of labor amongst campuses Communication and Cooperation Intercampus Allows for optimal efficiency in the lab Intracampus Division of labor effectively Increased the amount of new ideas Increased productivity Size of the school is not important
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We had a flippin’ good time!!!
But above all… We had a flippin’ good time!!! We’d be happy to take questions. Thank you. Note: Might want to add acknowledgements here, team members, iGEM, funding, etc.
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E.HOP: A Bacterial Computer to Solve the Pancake Problem
Collaborators at MWSU: Marian Broderick, Adam Brown, Trevor Butner, Lane Heard, Eric Jessen, Kelly Malloy, Brad Ogden Support: Davidson College HHMI NSF Genome Consortium for Active Teaching James G. Martin Genomics Program
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Extra Slides >>>
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Modeling 384 Stacks of 4 Pancakes
2n (n!) = X Combinations 24 (4!) = 384 Combinations
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Modeling 384 Stacks of 4 Pancakes
* * Build one representative of each family * * * *
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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
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8 vertices, each with degree 3
1 2 -1 2 1 -2 -1 -2 -2 1 -2 -1 2 -1 2 1 P2: 2 Burnt Pancakes 8 vertices, each with degree 3 Flip length 1 Flip length 2
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8 vertices, each with degree 3
1 2 -1 2 -1 -2 1 -2 -2 -1 -2 1 2 1 2 -1 1 1 -2 2 -1 1 2 -1 2 1 -2 -1 -2 -2 1 -2 -1 2 -1 2 1 P2: 2 Burnt Pancakes 8 vertices, each with degree 3
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8 vertices, each with degree 3
1 2 -1 2 -1 -2 1 -2 -2 -1 -2 1 2 1 2 -1 3 2 1 -2 2 -1 1 2 -1 2 1 -2 -1 -2 -2 1 -2 -1 2 -1 2 1 B2: 2 Burnt Pancakes 8 vertices, each with degree 3
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