Davidson – Missouri Western Foundational Advances in Biology and the Knapsack Problem
What is the knapsack problem? ? 2 kg 12 kg 15 kg 1 kg 4 kg
What is the knapsack problem? 2 kg 12 kg 15 kg 1 kg
Why is it so hard to solve? ? 35 kg 49 kg 33 kg 31 kg 87 kg 30 kg 47 kg 25 kg 26 kg 33 kg
Why is it so hard to solve? 31 kg 87 kg 30 kg 26 kg
Choosing Items with the Cre/Lox System FP TetA n FP TetA n FP TetA n EXCISION INVERSION EXCISION
Excision Cre Cre FP TetA n FP TetA n FP TetA n Cre Cre
Excision Cre Cre FP TetA n FP TetA n FP TetA n Cre Cre
Excision Cre Cre FP TetA n FP TetA n Cre Cre
Excision Cre Cre FP TetA n FP TetA n Cre Cre
Inversion Cre Cre FP TetA n FP TetA n FP TetA n Cre Cre
Inversion Cre Cre FP TetA n FP TetA n FP TetA n Cre Cre
Inversion Cre Cre FP TetA n TetA n FP FP TetA n Cre Cre
Inversion Cre Cre FP TetA n TetA n FP FP TetA n Cre Cre
Encoding Item Weights with Tet Pumps TetA Pumps
Too Few Pumps Too Many Pumps Just Right
Too Few Pumps Too Many Pumps Just Right
Too Many Pumps DEATH # of TetA Pumps DEATH Too Few Pumps Tetracycline Concentration
Cre RFP TetA
Building a Biological Computer lox sites = choice Tet R = weight tetA RFP nFP = object
Building a Biological Computer Lox sites = choice Tet R = weight tetA RFP nFP = object
Building a Biological Computer Lox sites = choice Tet R = weight tetA RFP nFP = object
Tools • The Oligator • Veripart • Construct Simulator • The Optimus • Knapsack Educational Game
The Oligator • Assists in de novo part construction • Designs oligos for annealing
• Assists in the analysis of sequence data • Compares raw sequence to Registry • Outputs matches to Registry parts
Construct Simulator • Simulates Cre action on Knapsack constructs • Demonstrates recombination of lox sites • Outputs fluorescent protein expression
Codon Optimization • Tet(A) production = object weight – optimized Tet(A) = heavy object – deoptimized Tet(A) = light object – wt Tet(A) = intermediate object
The Optimus • Uses codon bias to alter DNA sequence for a given polypeptide • Frequently used codons (optimization) or rarely used codons (deoptimization) are selected • Possible mechanism to regulate gene expression
Expanding the Cre/lox System • Built 11 novel lox sites that exhibit mutually exclusive recombination F R
Characterization of recombination • 21 possible lox combinations • Each lox variant is color coded above
Expanding the Cre/lox System • 11 new lox variants added to registry • Characterizing valuable foundational tool for the SB community • Tested pBad‐RBS‐Cre: wrong part • Rebuilt, sequence verified pBad‐RBS‐Cre, and added to registry pBAD Cre RBS
Foundational Issues • Designed & modeled Knapsack Problem computer • Initial testing revealed anomalies • Two options: 1. Ignore 2. Investigate • We chose to explore foundational issues
Five Hard Truths for Synthetic Biology 1. Many of the parts are undefined 2. The circuitry is unpredictable 3. The complexity is unwieldy 4. Many parts are incompatible 5. Variability crashes the system Kwok, Roberta. 2010. Five Hard Truths for Synthetic Biology. Nature. 463:288‐290
Five Hard Truths for Synthetic Biology 1. Many of the parts are undefined 2. The circuitry is unpredictable 3. The complexity is unwieldy 4. Many parts are incompatible 5. Variability crashes the system Kwok, Roberta. 2010. Five Hard Truths for Synthetic Biology. Nature. 463:288‐290
Observation #1 Circuitry is Unpredictable RBS RFP RBS TetA RFP+Tet Tet+RFP RBS TetA RBS RFP RBS RFP RFP
Observation #1 Circuitry is Unpredictable RFP alone RFP alone RFP 1 st RFP 1 st RFP 2 nd RFP 2 nd
Observation #2 Many Parts are Incompatible AMP + AMP TET + IPTG TET IPTG
Observation #3 Many of the parts are undefined Fluorescence Measured Over a Period of Time in Water and Broth pLac‐RBS‐RFP 144 Water LB Broth 120 96 AU 72 48 Water LB 24 0 pLac‐RBS‐RFP pLac‐RBS‐RFP Various Constructs (allowed to sit in water or broth) 8:30am 10:00am 12:00pm 2:00pm
Observation #4 The Circuitry is Unpredictable Color Pink Variation Red
Observation #4 The Circuitry is Unpredictable Red • DNA Sequence identical Pink
Observation #4 The Circuitry is Unpredictable Pink Clone Red Clone Red Clone Pink Clone
Observation #5 The Circuitry is Unpredictable C B Ring A
C A B Ring
Accomplishments • Designed biological computer for Knapsack problem • Developed 5 software tools for SB • Built library of 11 new lox sites • Built 42 basic and composite parts • Rebuilt Cre • Documented foundational issues
Foundational Advances • Evidence of transcriptional terminator in TetA • Quantified interference with fluorescence – Tetracycline – Water vs. media • Possible epigenetic regulation • Bacterial community dynamics
Acknowledgements • NSF Grants DMS 0733952 and 0733955 • HHMI Grant 52006292 • Davidson College Faculty Study and Research Grant, Davidson Research Initiative, and James G. Martin Genomics Program • Missouri Western State University Foundation, Academic and Student Affairs, and Summer Research Institute • Genome Consortium for Active Teaching (GCAT) • David Bikard from 2007 Paris iGEM Team
Davidson – Missouri Western Foundational Advances in Biology and the Knapsack Problem Kelia Alfred, Stacey Holle, Bridget Janssen, Curtiss Lane, Steph Meador, Bri Pearson, Jamela Peterson, Anvi Raina, Nitya Rao, Eugene Shiu, Tom Shuman, Jeff Stevens, Stephen Streb, Jeska Testa, Danielle Wagner
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