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DUKE iGEM. Aakash Indurkhya, Peter Fan, and Alyssa Ferris. introduction. Designing for the Future. We identified a need for custom made synthetic biological parts. This gives more power and control over networks than naturally present biological parts. introduction.

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DUKEiGEM

Aakash Indurkhya, Peter Fan, and Alyssa Ferris


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introduction

Designing for the Future

We identified a need for custom madesynthetic biological parts.

This gives more power and control over networks than naturally present biological parts


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introduction

The Future of Synthetic Biology

Embryonic development uses a natural genetic toggle switches

Variations in the toggle switch hold promisefor research toward a cure for type-1 diabetes


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Zinc fingers

Design

Characterization

Experimental


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Zinc Fingers

Zinc fingers as transcription factors

  • We are creating a library of synthetic repressor-promoter pairs

  • Zinc fingers are strong DNA binding domains

    Multi-finger arrays can act as repressors through steric hindrance of RNA Polymerase.


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Zinc Fingers

Zinc Finger Arrays

α (or recognition) helices bind to 3 bpof DNA with high affinity

Developing assembly methods allow custom made TFs.


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Zinc Fingers

Context-Dependent Assembly (CoDA)

ZFA Assembly Methods

Pre-screened arrays

Sander et al, 2011


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Design

Characterization

Experimental

Conclusion


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Design

The original Genetic Toggle Switch

Gardner et al, 2000


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Design

Characteristics of Toggle Switches

  • Bi-stability

  • Reporter or marker structural genes

  • Repressible Constitutive Promoters

  • Low Basal Transcriptional Noise

Image taken from: http://parts.mit.edu/igem07/index.php/Tokyo/sunaba2


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Design

Controller Mechanism

Split the Toggle Switch into two plasmids:

  • One containing [double-repression] activation of inducible promoters

  • The other accounting for bi-stability in gene expression

Reporter Gene 1

Reporter Gene 2


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Design

Network Overview

Controller Plasmid


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Characterization

Experimental

Conclusion


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Characterization

Graphical Representation

  • Multiple repression system serves to activate promoters

  • This design accounts for:

  • Reduced transcriptional noise

  • Activation threshold


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Characterization

Graphical Representation

  • Zinc Finger transcriptional repressors forms the core of the Toggle Switch Controller

  • This allows for inputs and outputs to be adjusted on demand


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Characterization

Graphical Representation

  • Negative Feedback Loops

  • Bi-stability

  • This design accounts for:

  • The toggling ability for the network.

  • Easy to determine network success

    • CFP: Blue

    • YFP: Yellow


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Characterization

Analogous Representation

Method of communication between remote and TV stays the same

User inputs and system outputs are based on desired outcome and response values


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Characterization

  • No inducers added

Gene Expression System

Time (minutes)


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Characterization

  • Insufficient addition of inducer A (or B)

Gene Expression System

Time (minutes)


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Characterization

  • Sufficient addition of inducer A (or B)

Gene Expression System

Time (minutes)


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Experimental

Conclusion


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Experimental

Selection of Zinc Finger Arrays


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Experimental

Computational Results


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Experimental

Experimental Characterization

Bacterial-two-hybrid assay

  • Standardized for 3-finger array characterization

  • Activator domain taken from eukaryotic system

  • Measure concentration of reporter gene

Maeder et al, 2009


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Experimental

Bacterial Two-Hybrid (B2H) Assay

Modified version from Wright et al, 2006


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Experimental

B2H Results

  • Long assay with tedious steps

  • Completed with inconclusive results

  • The construction of B2H reporter strain has several opportunities for error


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Experimental

Construction: CPEC

  • Initial PCR adds overlappingregions

  • Second PCR attaches the insert to the vector

Use CPEC to replace tedious construction steps

http://www.nature.com.proxy.lib.duke.edu/nprot/journal/v6/n2/full/nprot.2010.181.html


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Future Work:

In the coming weeks:

We plan to test CPEC as a means to construct the B2H reporter strain

  • Experimental characterization completed very quickly

    Our network fragments are being synthesizedde novo

    - FACS analysis and Fluorescence microscopy

    - Confirm network success


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Conclusions

Conclusions

  • We have

  • Developed a new screen and characterization method for zinc fingers.

  • Designed and produced 9 custom made zinc finger repressors as BioBricks

  • Identified a use for the new TFs in an improvement to the genetic toggle switch.

  • Engineered and modeled the genetic toggle switch controller

  • Propose a more efficient construction process for the bacterial-two-hybrid assay.


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Conclusions

How this fits in:

Engineering

Two plasmid Toggle Switch Controller

Custom made synthetic zinc finger repressors

Apply new ideas

Try something new

Improve ideas


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Conclusions

Team Members

NCSSM Students

Undergraduate

Aakash Indurkhya

Peter Fan

Kevin Chien

Alyssa Ferris


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Conclusions

Acknowledgements

  • We would like to thank the Tian Lab for hosting our research and our sponsors at the NCSSM.

  • Mentors and Advisors: Dr. Tian, Dr. Halpin, Dr. Buchler, Dr. Gersbach, Mr. Gotwals, Dr. Sheck, Ms. Ma, and Mr Tang.