A Genetic Toggle Switch Using DNA Recombinases Michigan Synthetic Biology Team 2012
Switch Design and Modeling Mike
Contemporary Bistable Switch
Disadvantages • The state is actively maintained • Sensitive • Complexity Lou et al., Molecular Systems Biology (2010) 6 (350)
Switch Design
Switch Design
Switch Design
Switch Design
Switch Design
Advantages • The state is passively maintained • Heritable • Robust • Simple Recombinase switch shown to be stable over 100 generations Bonnet et al., PNAS (2012)
Modeling • We modeled our system using a set of mass action ODE's • Qualitatively explored the functionality of our circuit • Analytically Solved for our Steady State
Switching, Leakage, Decay
Switching, Leakage, Decay
Positive Feedback Loop
Characterization Josh
Fluorescent Reporter (K137058) CalTech 2008
Fluorescent Reporter (K137058) CalTech 2008
Asymmetrical Digest Reporter (K880003) Michigan 2012 123bp 136bp 357bp
Asymmetrical Digest Reporter (K880003) Michigan 2012 123bp 256bp 238bp
Asymmetrical Digest Reporter (K880003) Michigan 2012 K880003 + FimE K880003 FimE 400bp 300bp Unflipped Flipped 200bp 100bp
Inverted Repeats in the Natural fimS region Stationary Mobile Mobile Stationary IRL IRR Stationary Mobile Mobile Stationary IRL IRR
Inverted Repeat Orientations IRL IRR IRL IRR IRR IRL
Asymmetrical Digest Reporter (K880001) Michigan 2012 123bp 136bp 357bp
Asymmetrical Digest Reporter (K880001) Michigan 2012 123bp 256bp 238bp
Asymmetrical Digest Reporter (K880001) Michigan 2012 K880001 K880001 K880001 K880001 + + + FimE FimE FimE 400bp Unflipped 400bp 300bp Flipped Unflipped 300bp 200bp Flipped 200bp 100bp 100bp
Fluorescent Reporter (K880002) Michigan 2012
Natural fimS accessory sites Roesch and Bloomfield Molecular Microbiology (1998) 27(4)
HbiF Characterization Ben
HbiF Design
HbiF Results - TEM HbiF expressing cells Untransformed control 500nm 500nm
Human Practice Corey
Goal Advocate and promote the field of synthetic biology to a younger audience, particularly high school students, through a peer mentor role.
Plan Collaborate with a campus organization, Women in Science & Engineering (WISE) at their annual day-camp to advocate synthetic biology . 4 Activities - • College panel lecture • 2 Day pGLO experiment • Introduction to iGEM • Survey/feedback session.
WISE Camp College panel lecture 2 Day pGLO experiment Introduction to iGEM Survey/feedback session.
Survey/Feedback Results >90% of the students expressed interest in getting involved with iGEM-HS ~90% will spread the word to their family, friends, and teacher ~78% students (36/46) heard about synthetic biology **all data based on a population 48 students
High School Students Two high school students from China volunteered 5 weeks of their summer working with the iGEM team, particularly cloning steps for moving our parts to submission plasmids Ivor Ocean
Future Direction • Collaborate with local Skyline High School • We plan on continued collaboration with WISE
Conclusion Mike
Our Accomplishments... • We helped to identify possible issues when working with the fim recombinase system.
Reporter Parts BBa_K880001 Asymmetrical Digest Reporter BBa_K880002 GFP fluorescent reporter
Our Accomplishments... • We introduced a new unidirectional recombinase HbiF to the registry and tested it BBa_K880000 HbiF Recombinase BBa_K880004 LVA Tag in RFC25
Our Accomplishments... • Our Human Practices project introduced synthetic biology to over 50 high school students around the country.
Looking Forward Build a better bi-stable switch: LRP and IHF sites Positive Feedback Loops Degradation Tags Higher-Level Applications: Biological Memory Modules Computational Circuits
Acknowledgements Our Advisors: Dr. Marcus Ammerlaan and Jeremy Minty
Thank You!
Appendix
Appendix
Appendix
Appendix
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