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![Page 1: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/1.jpg)
Living Hardware to Solve the Hamiltonian Path Problem
Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer
Students: Oyinade Adefuye, Will DeLoache, Jim Dickson, Andrew Martens, Amber Shoecraft, and Mike Waters
![Page 2: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/2.jpg)
The Hamiltonian Path Problem
![Page 3: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/3.jpg)
• Millennium Problem • P=NP?• Brute Force required
Does a Hamiltonian Path exist in this graph?
Computational Complexity
![Page 4: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/4.jpg)
Why Should We Use Bacteria?
Adleman LM (1994). Science 266 (11): 1021-1024.
VS.
![Page 5: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/5.jpg)
Flipping DNA with Hin/hixC
![Page 6: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/6.jpg)
Using Hin/hixC to Solve the HPP
![Page 7: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/7.jpg)
Using Hin/hixC to Solve the HPP
hixC Sites
![Page 8: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/8.jpg)
Using Hin/hixC to Solve the HPP
![Page 9: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/9.jpg)
Using Hin/hixC to Solve the HPP
![Page 10: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/10.jpg)
Using Hin/hixC to Solve the HPP
Solved Hamiltonian Path
![Page 11: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/11.jpg)
What Genes Can Be Split?
GFP before hixC insertion
![Page 12: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/12.jpg)
What Genes Can Be Split?
GFP displaying hixC insertion point
![Page 13: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/13.jpg)
Gene Splitter Software
Input Output
1. Gene Sequence
2. Where do you want your hixC site?
3. Pick an extra base to avoid a frameshift
1. Generates 4 Primers (optimized for melting temperature).
2. Biobrick ends are added to primers.
3. Frameshift is eliminated.
![Page 14: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/14.jpg)
Gene-Splitter Output
Note: Oligos are optimized for melting temperatures.
![Page 15: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/15.jpg)
Use GFP to Split RFP
Green Fluorescent Protein Red Fluorescent Protein
![Page 16: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/16.jpg)
Can We Detect A Solution?
![Page 17: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/17.jpg)
Elements in the shaded region can be arranged in any order.
True Positives
Number of True Positives = (Edges-Nodes+1)! * 2(Edges-Nodes+1)
![Page 18: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/18.jpg)
False Positives
Extra Edge
![Page 19: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/19.jpg)
False Positives
PCR Fragment Length
PCR Fragment Length
![Page 20: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/20.jpg)
0
0.25
0.5
0.75
1
4/6 6/9 7/12 7/14
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
4/6 6/9 7/12 7/14
Detection of True Positives
Tot
al #
of P
ositi
ves
# of
Tru
e P
ositi
ves
÷
Tot
al #
of P
ositi
ves
# of Nodes / # of Edges
# of Nodes / # of Edges
![Page 21: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/21.jpg)
k = actual number of occurrences
λ = expected number of occurrences
λ = m plasmids * # solved permutations of edges ÷ # permutations of edges
Cumulative Poisson Distribution:
How Many Plasmids Do We Need?
1 mL of culture = 10 cells 9
P(# of solutions ≥ k) = 1 - ∑k-1
X=0
_____e-λ λX!
x.
![Page 22: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/22.jpg)
Starting ArrangementP
roba
bilit
y of
HP
P S
olut
ion
Number of Flips
4 Nodes & 3 Edges
![Page 23: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/23.jpg)
Where Are We Now?
![Page 24: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/24.jpg)
First Bacterial Computer
Starting Arrangement
![Page 25: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/25.jpg)
First Bacterial Computer
Starting Arrangement
Solved Arrangement
![Page 26: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/26.jpg)
Future Directions
Split additional genes:
Solve other problems such as the Traveling Salesperson Problem:
Make more complex graphs:
![Page 27: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/27.jpg)
Living Hardware to Solve the Hamiltonian Path Problem
Collaborators at MWSU:
Support: Davidson College The Duke Endowment
HHMI NSF Genome Consortium for Active Teaching James G. Martin Genomics Program
Dr. Todd Eckdahl, Dr. Jeff Poet, Jordan Baumgardner,Tom Crowley, Lane H. Heard, Nickolaus Morton, Michelle Ritter, Jessica Treece, Matthew Unzicker, Amanda Valencia
Additional Thanks: Karen Acker, Davidson College ‘07
![Page 28: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/28.jpg)
Extra Slides
![Page 29: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/29.jpg)
Traveling Salesperson Problem
![Page 30: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/30.jpg)
Processivity
•The nature of our construct requires a stable transcription mechanism that can read through multiple genes in vivo
•Initiation Complex vs. Elongation Complex
•Formal manipulation of gene expression (through promoter sequence and availability of accessory proteins) is out of the picture
Problem:
Solution : T7 bacteriophage RNA polymerase
• Highly processive single subunit viral polymerase which maintains processivity in vivo and in vitro
![Page 31: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/31.jpg)
Path at 3 nodes / 3 edges HP- 1 12 23
1 2
3TT
![Page 32: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/32.jpg)
Path at 4 nodes / 6 edges HP-1 12 24 43
2
34
1
TT
![Page 33: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/33.jpg)
Path 5 nodes / 8 edges HP -1 12 25 54 43
2
34
1
TT
5
![Page 34: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/34.jpg)
Path 6 nodes / 10 edgesHP-1 12 25 56 64 43
2
34
1
TT
56
![Page 35: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/35.jpg)
Path 7 nodes / 12 edgesHP-1 12 25 56 67 74 43
2
34
1
TT
56
7
![Page 36: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/36.jpg)
More Gene-Splitter Output
![Page 37: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/37.jpg)
Promoter Tester
• RBS:Kan:RBS:Tet:RBS:RFP• Tested promoter-promoter tester-pSBIA7 on varying concentration plates
Kanamycin Tet Kan-Tet
50 50 50 / 50
75 75 75 / 75
100 100 100 / 100
125 125 125 / 125
•Used Promoter Tester-pSB1A7 and Promoter Tester-pSB1A2 without promoters as control
•Further evidence that pSB1A7 isn’t completely insulated
![Page 38: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/38.jpg)
Promoters Tested
•Selected “strong” promoters that were also repressible from biobrick registry
•ompC porin (BBa_R0082)
•“Lambda phage”(BBa_R0051)
•pLac (BBa_R0010)
•Hybrid pLac (BBa_R0011)
•None of the promoters “glowed red”
•Rus (BBa_J3902) and CMV (BBa_J52034) not the parts that are listed in the registry
![Page 39: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/39.jpg)
•Determined hixC site insertion at AA 125 in each monomer subunit-AA 190 is involved in catalysis
-AA 195 and 208 are involved in Mg2+ binding
-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT -Substitution of AA 210 (conserved) reduced enzyme activity -AA 166 serves to catalyze reactions involving ATP
-AA 44 is involved in ATP binding
-AA 60 is involved in orientation of AA 44 and ATP binding
-We did not consider any Amino Acids near the N or C terminus
-Substitution of AA 190 caused 650-fold decrease in enzyme activity
-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary
structure and therefore the hydrogen bonding ability of KNTase)
•Did not split
Splitting Kanamycin Nucleotidyltransferase
![Page 40: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/40.jpg)
Plasmid Insulation
• “Insulated” plasmid was designed to block read-through transcription
•Read-through = transcription without a promoter
•Tested a “promoter-tester” construct
•RBS:Kan:RBS:Tet:RBS:RFP
•Plated on different concentrations of Kan, Tet, and Kan-Tet plates
•Growth in pSB1A7 was stunted
•No plate exhibited cell growth in uninsulated plasmid and cell death in the insulated plasmid
![Page 41: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/41.jpg)
Tetracycline Resistance Protein
•Did not split
•Transmembrane protein
•Structure hasn’t been crystallized
•determined by computer modeling
•Vital residues for resistance are in transmemebrane domains (efflux function)
•HixC inserted a periplasmic domains AA 37/38 and AA 299/300
•Cytoplasmic domains allow for interaction with N and C terminus
![Page 42: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/42.jpg)
Splitting Cre Recombinase
![Page 43: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/43.jpg)
What Genes Can Be Split?
GFP before hixC insertion GFP displaying hixC insertion point
![Page 44: Living Hardware to Solve the Hamiltonian Path Problem Professors: Dr. Malcolm Campbell and Dr. Laurie Heyer Students: Oyinade Adefuye, Will DeLoache, Jim.](https://reader030.fdocuments.in/reader030/viewer/2022013011/56649e0e5503460f94af7750/html5/thumbnails/44.jpg)
Gene Splitter Software