Support is gratefully acknowledged from NSF UBM grants DMS-0733952 & 0733955, HHMI grant #52006292, Davidson College Faculty Study and Research Grant, DRI, the Davidson College Martin Genomics Program, the Missouri Western Foundation, Missouri Western Academic and Student Affairs, Missouri Western SRI, GCAT, and David Bikard.

We developed a genetic circuit that would allow bacterial computers to solve the knapsack problem. Investigation of unexplained observations led to foundational advances in codon optimization, cre-lox characterization, and gene expression. We also designed several software tools.

Our accomplishments in foundational advances included a design for a biological computer for the Knapsack problem, software tools, 11 new lox sites, investigations into unexplained observations in gene expression, built 42 basic and composite parts, and identification of a transcriptional terminator in the TetA gene.

For the design of optimized and deoptimized sequences, we used Optimus and Oligator. We cut the TetA gene using natural restriction enzyme sites and inserted our newly designed sequence. RFLP was used to select candidate clones for our optimized and deoptimized versions of the TetA gene.

Inversion

Excision

In order to randomly "select objects" for the knapsack problem, we used the Cre-lox recombination method of excision and inversion. We needed a set of variant lox sites to build constructs that would yield different subsets of survival and fluorescence. Overall, we engineered 11 new lox sites with mutations in the 8 bp region.

VeriPart assists in the analysis of sequence data by comparing raw sequences to the registry.

The Oligator assists in de novo part construction and designs oligos for annealing between 20 and 20,000 base pairs.

The Optimus uses codon bias to alter DNA sequence for a given polypeptide based on the RCBS-PC or the CAI formula. Selects frequently used codons (optimization) or rarely used codons (deoptimization).

SimuLox simulates Cre action on Knapsack constructs, demonstrates recombinations of lox sites, and outputs fluorescent protein expression.

The knapsack problem is an NP-complete problem that asks: given a capacity and different weighted items, does there exists a subset of the items for which the sum of their weights is equal to that of the capacity?

Biological construction of the Knapsack problem:

The design and construction of a Knapsack biological computer led to several unexplained observations. Ongoing investigation promises Foundational Advances in the measurement of gene expression.

Our research led us to a good design concept for biological implementation of the Knapsack problem. However, initial construction efforts revealed several unexplained observations. We realized that we could take either of two paths:

1.  Ignore Unexplained Observations 2.  Investigate Foundation Issues

We chose to explore the foundational issues.

Observation #1 - Order of RFP and TetA genes in an Operon Affects RFP Expression.

FoundationalAdvancesandtheKnapsackProblemKeliaAlfred6,StaceyHolle5,BridgetJanssen4,CurtissLane4,StephMeador2,BriPearson1,JamelaPeterson1,AnviRaina1,

NityaRao1,EugeneShiu2,TomShuman1,JeffStevens5,StephenStreb2,JeskaTesta4,DanielleWagner4,LaurieHeyer2,3,JeffPoet3,5,ToddEckdahl3,4,A.MalcolmCampbell1,3

1Biology Department, 2Mathematics Department, Davidson College; 3Genome Consortium for Active Teaching (GCAT); 4Biology Department, 5Mathematics Department, Missouri Western State University; 6North Carolina A & T State University.

Observation #2 - RFP expression reduced when TetA is required.

Observation #3 - RFP expression increased in water.

Observation #4 - Two RFP expression clones cause different fluorescence levels despite identical DNA sequences.

Observation #5 - RFP expression clone has variation in fluorescence on plates. Differences not explained by mutations.

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