Back to the Basics

Purdue iGEM 2013

The Biomakers

Back to the Basics

• What Do We Mean?

• Complex Projects

• Lack of Fundamental Control

The Taguchi MethodCheaper, Faster Characterization

Explanation of Problem

• Increase Robustness of Genetic Circuits

• Assess with Factorial Experiment

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

Comparison of Methods

•Partial-Factorial Method

•Fewer Combinations

Taguchi Method

• All Possible Combinations• Often Not Feasible

Full Factorial

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

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)

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 DesignIncreasing 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

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

Conclusion

• Promoter Submitted to Registry

• Implementation

• Increase Reliability

Future Work

• Compare Biobrick BCDs to Original BCDs

• Submit BCDs to iGEM HQ

Standardized DatasheetsRevolutionizing 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: 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

Sponsors

Questions?