Living Hardware to Solve the Hamiltonian Path Problem Faculty: Drs. Malcolm Campbell, Laurie Heyer, Karmella Haynes Students: Oyinade Adefuye, Will DeLoache, Jim Dickson, Andrew Martens, Amber Shoecraft, and Mike Waters Faculty: Drs. Todd Eckdahl, Jeff Poet Students: Jordan Baumgardner, Tom Crowley, Lane H. Heard, Nickolaus Morton, Michelle Ritter, Jessica Treece, Matthew Unzicker, Amanda Valencia

The Hamiltonian Path Problem

Advantages of Bacterial Computation SoftwareHardwareComputation

Advantages of Bacterial Computation SoftwareHardwareComputation

Advantages of Bacterial Computation SoftwareHardwareComputation

Computational Complexity Non-Polynomial (NP) No Efficient Algorithms

Computational Complexity Cell Division Non-Polynomial (NP) No Efficient Algorithms

Flipping DNA with Hin/hixC

Using Hin/hixC to Solve the HPP

hixC Sites Using Hin/hixC to Solve the HPP

Solved Hamiltonian Path

How to Split a Gene RBS Promoter Reporter Detectable Phenotype

How to Split a Gene RBS Promoter Reporter hixC RBS Promoter Repo- rter Detectable Phenotype Detectable Phenotype ?

Gene Splitter Software InputOutput 1. Gene Sequence (cut and paste) 2. Where do you want your hixC site? 3. Pick an extra base to avoid a frameshift. 1.Generates 4 Primers (optimized for Tm). 2. Biobrick ends are added to primers. 3. Frameshift is eliminated.

Gene-Splitter Output Note: Oligos are optimized for Tm.

Can We Detect A Solution?

Starting Arrangement Probability of HPP Solution Number of Flips 4 Nodes & 3 Edges

Elements in the shaded region can be arranged in any order. True Positives Number of True Positives = (Edges-(Nodes-1))! * 2 (Edges-Nodes+1)

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? P(# of solutions ≥ k) = k = m = 10,000, ,000, ,000, ,000, ,000, ,000,000, Probability of at least k solutions on m plasmids for a 14-edge graph

False Positives Extra Edge

False Positives PCR Fragment Length

Detection of True Positives Total # of Positives # of True Positives ÷ Total # of Positives # of Nodes / # of Edges

Building a Bacterial Computer

Splitting Reporter Genes Green Fluorescent ProteinRed Fluorescent Protein

Splitting Reporter Genes Green Fluorescent Protein Red Fluorescent Protein

3-Node Graphs Graph AGraph B

HPP Constructs Graph A Constructs: Graph B Construct: HPP 0 HPP-A 0 HPP-A 1 HPP-A 2 HPP-B 1 Positive Control Construct: Graph B Graph A

Green Fluorescence Double Fluorescence HPP 0 T7 RNAP

HPP-A 0 T7 RNAP Green Fluorescence Yellow Fluorescence Double Fluorescence HPP 0 T7 RNAP

Fluorometer Measurements GFP Excitation Spectra for 4 HPP Constructs (at an Emission Wavelength of 515nm) 450 nm chosen as excitation wavelength to measure GFP

Fluorometer Measurements RFP Excitation Spectra for 4 HPP Constructs (at an Emission Wavelength of 608nm) 560 nm chosen as excitation wavelength to measure RFP

Normalized Fluorometer Measurements ConstructFluorescent Color on UV Box GreenRed pLac-RBS-RFPRed7263 pLac-RBS-RFP-RBS-GFPRed pLac-GFP1-hixC-GFP2Green1360 pLac-RBS-RFP1-hixC-RFP2None0147 pLac-RBS-GFP1-hixC-RFP2None112 pLac-RBS-RFP1-hixC-GFP2None132 HPP-B 0 Green7218 HPP-A 0 Yellow HPP-A 1 Red1143 HPP-A 2 None113 HPP-B 1 Hybrid green153

Flipping Detected by Phenotype HPP-A 0 HPP-A 1 HPP-A 2

Flipping Detected by Phenotype Hin-Mediated Flipping HPP-A 0 HPP-A 1 HPP-A 2

Flipping Detected by PCR HPP-A 0 HPP-A 1 HPP-A 2 UnflippedFlipped

Pending Experiments Test clonal colonies that contain flipped HPP and have the solution sequenced. Perform a false-positive screen for HPP-B 1 Split 2 antibiotic resistance genes using a reading frame shift just after the RBS Solve larger graphs Solve the Traveling Salesperson Problem

Living Hardware to Solve the Hamiltonian Path Problem Support: Davidson College Missouri Western State University The Duke Endowment HHMI NSF Genome Consortium for Active Teaching James G. Martin Genomics Program Thanks to: Karen Acker, Davidson College ‘07

Extra Slides

Traveling Salesperson Problem

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

Path at 3 nodes / 3 edges HP T T

Path at 4 nodes / 6 edges HP T T

Path 5 nodes / 8 edges HP T T 5

Path 6 nodes / 10 edges HP T T 56

Path 7 nodes / 12 edges HP T T 5 6 7

Promoter Tester RBS:Kan:RBS:Tet:RBS:RFP Tested promoter-promoter tester-pSBIA7 on varying concentration plates KanamycinTetKan-Tet / / / / 125 Used Promoter Tester-pSB1A7 and Promoter Tester-pSB1A2 without promoters as control Further evidence that pSB1A7 isn’t completely insulated

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

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

What Genes Can Be Split? GFP before hixC insertionGFP displaying hixC insertion point

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

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

Splitting Cre Recombinase

More Gene-Splitter Output

Gene Splitter Software