A Genetic Toggle Switch Using DNA Recombinases Michigan Synthetic - - PowerPoint PPT Presentation

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a genetic toggle switch using dna recombinases
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A Genetic Toggle Switch Using DNA Recombinases Michigan Synthetic - - PowerPoint PPT Presentation

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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.,

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A Genetic Toggle Switch Using DNA Recombinases

Michigan Synthetic Biology Team 2012

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Switch Design and Modeling Mike

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Contemporary Bistable Switch

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Disadvantages

  • The state is actively maintained
  • Sensitive
  • Complexity

Lou et al., Molecular Systems Biology (2010) 6 (350)

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Switch Design

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Switch Design

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Switch Design

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Switch Design

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Switch Design

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Advantages

  • The state is passively maintained
  • Heritable
  • Robust
  • Simple

Recombinase switch shown to be stable over 100 generations Bonnet et al., PNAS (2012)

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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
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Switching, Leakage, Decay

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Switching, Leakage, Decay

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Positive Feedback Loop

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Characterization Josh

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Fluorescent Reporter (K137058)

CalTech 2008

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Fluorescent Reporter (K137058)

CalTech 2008

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357bp

Asymmetrical Digest Reporter (K880003)

Michigan 2012

123bp 136bp

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238bp

Asymmetrical Digest Reporter (K880003)

Michigan 2012

123bp 256bp

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Asymmetrical Digest Reporter (K880003)

Michigan 2012

Unflipped Flipped

K880003 FimE K880003 + FimE

200bp 100bp 300bp 400bp

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Stationary Stationary Stationary Stationary Mobile Mobile Mobile Mobile

Inverted Repeats in the Natural fimS region

IRL IRL IRR IRR

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Inverted Repeat Orientations

IRL IRL IRR IRR IRR IRL

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357bp 123bp 136bp

Asymmetrical Digest Reporter (K880001)

Michigan 2012

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238bp 123bp 256bp

Asymmetrical Digest Reporter (K880001)

Michigan 2012

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Asymmetrical Digest Reporter (K880001)

Michigan 2012

Unflipped Flipped Unflipped Flipped 200bp 100bp 300bp 400bp 200bp 100bp 300bp 400bp

K880001 + FimE K880001 + FimE K880001 + FimE K880001

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Fluorescent Reporter (K880002)

Michigan 2012

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Natural fimS accessory sites

Roesch and Bloomfield Molecular Microbiology (1998) 27(4)

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HbiF Characterization Ben

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HbiF Design

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HbiF Results - TEM

HbiF expressing cells Untransformed control

500nm 500nm

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Human Practice Corey

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Goal

Advocate and promote the field of synthetic biology to a younger audience, particularly high school students, through a peer mentor role.

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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.
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WISE Camp

College panel lecture 2 Day pGLO experiment Introduction to iGEM Survey/feedback session.

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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

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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

Ocean Ivor

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Future Direction

  • Collaborate with local Skyline High School
  • We plan on continued collaboration with WISE
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Conclusion Mike

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Our Accomplishments...

  • We helped to identify possible issues when

working with the fim recombinase system.

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Reporter Parts

BBa_K880001 Asymmetrical Digest Reporter BBa_K880002 GFP fluorescent reporter

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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

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Our Accomplishments...

  • Our Human Practices project introduced

synthetic biology to over 50 high school students around the country.

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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

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Acknowledgements

Our Advisors:

  • Dr. Marcus Ammerlaan and Jeremy Minty
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Thank You!

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Appendix

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Appendix

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Appendix

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Appendix