Back to the Basics
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Transcript of Back to the Basics
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Back to the Basics
Purdue iGEM 2013
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The Biomakers
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Back to the Basics
• What Do We Mean?
• Complex Projects
• Lack of Fundamental Control
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The Taguchi MethodCheaper, Faster Characterization
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Explanation of Problem
• Increase Robustness of Genetic Circuits
• Assess with Factorial Experiment
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The Taguchi Method
Sources: (2013). IGCSE and GCSE Industry. Retrieved from http://greenfieldgeography.wikispaces.com/IGCSE+and+GCSE+Industry Venil, C. K., & Lakshmanapermalsamy, P. (2009). Taguchi Experiment Design.... Journal of Science & Technology
• Used in Industry– Maintain quality
• Our Project– Apply Taguchi to synthetic biology
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Comparison of Methods
•Partial-Factorial Method
•Fewer Combinations
Taguchi Method
• All Possible Combinations• Often Not Feasible
Full Factorial
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Experimental Design
• Parameters
• Orthogonal Arrays
• Response Variable: GFPFactors Promoter RBS Terminator E. coli Strains
Levels
Weak Weak Double Terminator BL21
Medium Medium ADH1 XL-1 Blue
Strong Strong T7 BM25.8
Taguchi Orthogonal Array
Promoter RBS Terminator Strain
Strong Prom. Weak RBS T7 XL1-Blue
Medium Prom. Strong RBS Double XL1-Blue
Medium Prom. Weak RBS ADH1 BL21
Medium Prom. Medium RBS T7 BM25.8
Weak Prom. Weak RBS Double BM25.8
Weak Prom. Strong RBS T7 BL21
Strong Prom. Medium RBS Double BL21
Strong Prom. Strong RBS ADH1 BM25.8
Weak Prom. Medium RBS ADH1 XL1-Blue
Venil, C. K., & Lakshmanapermalsamy, P. (2009). Taguchi experimental design for medium optimization…. Journal of Science & Technology
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Intended Data Analysis
• Determining GFP Expressed Per Cell– Growth curve– Coulter counter
• JMP Statistical Analysis
Actual DataHypothetical Data
0 1 2 3 4 5 6 7 80
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Growth Curve
BL21BM25.8XL-1 Blue
Time (hours)
OD6
00
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.80
1
2
3
4
5
6
7
Standard Curve
OD
Cell
# (1
07)
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Challenges
• Transformations– Competent cells– Altered growing conditions
• 3A Assembly– Not feasible
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Future Work
• Complete Assembly
• Measure GFP per Cell for All Combinations
• Compare Results of Taguchi vs. Full Factorial
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Bicistronic DesignIncreasing Reliability of Expression
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Explanation of Problem
• Variation in Protein Expression
• Increase Reliability of Genetic Constructs
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BCD Background
• Bicistronic Design
Sources:Mutalik, V., Endy, D., Guimaraes, J., Cambray, G., Lam, C., Juul, M., Tran, A., & Paull, M. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements . Nature, 10(4)
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Monocistronic Design
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Bicistronic Design
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Supporting Data
• Protein Expression Variability Decreased
• Increase Protein Expression Reliability
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Monocistronic Design Bicistronic Design
Sources:Mutalik, V., Endy, D., Guimaraes, J., Cambray, G., Lam, C., Juul, M., Tran, A., & Paull, M. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements . Nature, 10(4)
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Design
• BCD Parts Designed
• Golden Gate Assembly
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Design
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Methods
• Synthesized Constructs
• Placed Ptrc* into pSB1C3
• Growth Rate Assay
• Re-Assembled Constructs into pSB1C3
• Proof of Function
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Data
• Promoter Submitted to the Registry
• Growth Rate Assay
• Fluorescence Intensity
0 1 2 3 4 5 6 7 8 9 100
0.5
1
1.5
2
2.5 Growth Curve
ControlpSB1C3 pSB1C3+prtc*
Time (hours)
OD6
00
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Conclusion
• Promoter Submitted to Registry
• Implementation
• Increase Reliability
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Future Work
• Compare Biobrick BCDs to Original BCDs
• Submit BCDs to iGEM HQ
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Standardized DatasheetsRevolutionizing Characterization
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Explanation of Problem
• Lack of Characterization
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Explanation of Problem
• Lack of Characterization• Vague Requirements• Difficult to Judge
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Background Research
• Standardized Protocol• Researched Past Protocols• Found Emails, Sent Survey• Survey Results Changed Direction
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First Draft
• Prior Art– Drew Endy
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First Draft
• Prior Art– Drew Endy – Boston iGEM
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First Draft
• Prior Art– Drew Endy – Boston iGEM
• First Draft– Design
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Feedback
• Prior Art– Drew Endy – Boston iGEM
• First Draft– Design– Implementation
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Feedback
• Prior Art– Drew Endy – Boston iGEM
• First Draft– Design– Implementation
• Second Survey and Video Conferences • Ideas Generated
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Final Design and Feedback
• BostonU iGEM Visit• Final Draft
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Final Design and Feedback
• BostonU iGEM Visit• Final Draft• Third Survey and Feedback
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Conclusion and Future Work
• Implementation• Continue Collaboration• Bba_K1225000
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Human Practices
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Intro and Overview
• Outreach to Producers• Outreach to High School• Girl Scouts Workshop• Biomaker Bench
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High School Outreach
• Teaching modeling to AP Biology Teachers– Educating next generation of Synthetic Biologists
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Girl Scout Outreach
• Generate interest in STEM• Workshop Curriculum
– Lecture– Experiment– Scientific method
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Biomaker Bench
• Creating a Community Lab in Noblesville, IN– Business plan– Nonprofit organization
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Conclusion
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Accomplishments
• Statistical analysis method to fit synthetic biology
• Updated Drew Endy's Bicistronic Design Constructs
• Designed a Datasheet Standard for the Registry
• Submitted a New Promoter and 4 BCD Constructs
• Collaborated with over 75 iGEM Teams
• Performed Outreach to Local Farmers and Producers
• Taught High School Teachers About Modeling
• Worked with the Girls Scouts of America
• Community Lab Space in Noblesville, Indiana
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Attributions and Acknowledgements
Advisers: Purdue University: Dr. Jenna Rickus Dr. Fernandez Dr. Kari Clase Dr. Dilkes Soo Ha Dr. Akridge Janie Brennan Jen Kahn
Bindley Bioscience Center: Research Support: Rajitarun Madangopal Boston University iGEM Dr. Larisa Avramova Drew Endy and the Endy Lab Dr. Lake Paul Dr. Tony Pedley
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Sponsors
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Questions?