Purdue iGEM 2013
Back to the Basics
The Biomakers
Back to the Basics
• What Do We Mean?
• Complex Projects
• Lack of Fundamental Control
The Taguchi Method
Cheaper, Faster Characterization
Explanation of Problem
• Increase Robustness of Genetic Circuits
• Assess with Factorial Experiment
The Taguchi Method
• Used in Industry
– Maintain quality
• Our Project
– Apply Taguchi to synthetic biology
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
Comparison of Methods
Full
Factorial
• All Possible Combinations
• Often Not Feasible
Taguchi
Method
• Partial-Factorial Method
• Fewer Combinations
Experimental Design
Taguchi Orthogonal Array
• Parameters
Promoter
• Orthogonal Arrays
• Response Variable: GFP
Factors
Levels
RBS
Terminator
Strain
Strong Prom.
Weak RBS
T7
XL1-Blue
Medium Prom.
Strong RBS
Double
XL1-Blue
Medium Prom.
Weak RBS
ADH1
BL21
Medium RBS
T7
Weak RBS
Double
Medium Prom.
BM25.8
Promoter RBS
Terminator
Weak
Weak Prom.
Weak
Double
Terminator
BL21
Strong RBS
T7
Medium
Medium
Strong
Prom.
ADH1
Medium RBS
Double
XL-1 Blue
BL21
Strong
Strong
Prom.
Strong
T7 Strong RBS
ADH1
BM25.8
BM25.8
Weak Prom.
Weak Prom.
Medium RBS
E. coli Strains
ADH1
Venil, C. K., & Lakshmanapermalsamy, P. (2009). Taguchi experimental design for medium optimization…. Journal of Science & Technology
BM25.8
BL21
XL1-Blue
Intended Data Analysis
• Determining GFP Expressed Per Cell
– Growth curve
– Coulter counter
GrowthCurve
Curve
Standard
1.8 7
1.6
• JMP Statistical Analysis
6
1.4
5
Cell # (107)
OD600
1.2
1 4
BL21
0.8 3
BM25.8
0.6
XL-1 Blue
2
0.4
0.2
1
0 0
0 0
10.2
20.4
30.6
0.8
4
51
OD
Time (hours)
Hypothetical
Actual Data Data
1.2
6
1.4
7
1.6
8
1.8
Challenges
• Transformations
– Competent cells
– Altered growing conditions
• 3A Assembly
– Not feasible
Future Work
• Complete Assembly
• Measure GFP per Cell for All Combinations
• Compare Results of Taguchi vs. Full Factorial
Bicistronic Design
Increasing Reliability of Expression
Explanation of Problem
• Variation in Protein Expression
• Increase Reliability of Genetic Constructs
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)
Monocistronic Design
Bicistronic Design
Supporting Data
• Protein Expression Variability Decreased
• Increase Protein Expression Reliability
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)
Design
• BCD Parts Designed
• Golden Gate Assembly
Design
Methods
• Synthesized Constructs
• Placed Ptrc* into pSB1C3
• Growth Rate Assay
• Re-Assembled Constructs into pSB1C3
• Proof of Function
Data
• Promoter Submitted to the Registry
• Growth Rate Assay
• Fluorescence Intensity
Growth Curve
2.5
OD600
2
1.5
Control
1
pSB1C3
pSB1C3+prtc*
0.5
0
0
2
4
6
Time (hours)
8
10
Conclusion
• Promoter Submitted to Registry
• Implementation
• Increase Reliability
Future Work
• Compare Biobrick BCDs to Original BCDs
• Submit BCDs to iGEM HQ
Standardized Datasheets
Revolutionizing Characterization
Explanation of Problem
• Lack of Characterization
Explanation of Problem
• Lack of Characterization
• Vague Requirements
• Difficult to Judge
Background Research
•
•
•
•
Standardized Protocol
Researched Past Protocols
Found Emails, Sent Survey
Survey Results Changed Direction
First Draft
• Prior Art
– Drew Endy
First Draft
• Prior Art
– Drew Endy
– Boston iGEM
First Draft
• Prior Art
– Drew Endy
– Boston iGEM
• First Draft
– Design
Feedback
• Prior Art
– Drew Endy
– Boston iGEM
• First Draft
– Design
– Implementation
Feedback
• Prior Art
– Drew Endy
– Boston iGEM
• First Draft
– Design
– Implementation
• Second Survey and Video Conferences
• Ideas Generated
Final Design and Feedback
• BostonU iGEM Visit
• Final Draft
Final Design and Feedback
• BostonU iGEM Visit
• Final Draft
• Third Survey and Feedback
Conclusion and Future Work
• Implementation
• Continue Collaboration
• Bba_K1225000
Human Practices
Intro and Overview
•
•
•
•
Outreach to Producers
Outreach to High School
Girl Scouts Workshop
Biomaker Bench
High School Outreach
• Teaching modeling to AP Biology Teachers
– Educating next generation of Synthetic Biologists
Girl Scout Outreach
• Generate interest in STEM
• Workshop Curriculum
– Lecture
– Experiment
– Scientific method
Biomaker Bench
• Creating a Community Lab in Noblesville, IN
– Business plan
– Nonprofit organization
Conclusion
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
Attributions and Acknowledgements
Advisers:
Dr. Jenna Rickus
Dr. Kari Clase
Soo Ha
Janie Brennan
Jen Kahn
Purdue University:
Dr. Fernandez
Dr. Dilkes
Dr. Akridge
Bindley Bioscience Center:
Rajitarun Madangopal
Dr. Larisa Avramova
Dr. Lake Paul
Dr. Tony Pedley
Research Support:
Boston University iGEM
Drew Endy and the Endy Lab
Sponsors
Questions?