DUKE iGEM

Aakash Indurkhya, Peter Fan, and Alyssa Ferris

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

The Future of Synthetic Biology

Embryonic development uses a natural genetic toggle switches

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

introduction

Zinc fingers

Design

Characterization

Experimental

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.

Zinc Fingers

Zinc Finger Arrays

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

Zinc Fingers

Developing assembly methods allow custom made TFs.

ZFA Assembly MethodsContext-Dependent Assembly

(CoDA)

Pre-screened arrays Sander et al, 2011

Zinc Fingers

Characterization

Experimental

Conclusion

Design

The original Genetic Toggle Switch

Gardner et al, 2000

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

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

Design

Reporter Gene 1

Reporter Gene 2

Network Overview

Controller Plasmid

Design

Characterization

Experimental

Conclusion

Graphical Representation

• Multiple repression system serves to activate promoters

This design accounts for:• Reduced transcriptional noise• Activation threshold

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

Characterization

Graphical Representation

Negative Feedback Loops• Bi-stabilityThis design accounts for:• The toggling ability for

the network.• Easy to determine

network success• CFP: Blue • YFP: Yellow

Characterization

Analogous Representation

Characterization

Method of communication

between remote and TV stays the same

User inputs and system outputs are based on desired

outcome and response values

No inducers added

Time (minutes)

Gen

e Ex

pres

sion

Sys

tem

Characterization

Insufficient addition of inducer A (or B)G

ene

Expr

essi

on S

yste

m

Time (minutes)

Characterization

Sufficient addition of inducer A (or B)G

ene

Expr

essi

on S

yste

m

Time (minutes)

Characterization

Experimental

Conclusion

Selection of Zinc Finger Arrays

• BLASTn screen of E. coli genome for ZF binding siteScreen

• Generated by ZiFiT• Set for Context dependent assembly

Coding Sequences

• PDB models generated by SWISS-model and w3DNA

• MolDock algorithm => Free Energy ValuesCharacterization

Experimental

Computational Results

0 1 2 3 4 5 6 7 8 9

-600

-590

-580

-570

-560

-550

-540

-530

-520

MolDock Binding Affinity for Zinc Finger Transcription Factors

ZF1

ZF2

ZF3

ZF4

ZF5

ZF6

ZF7

ZF8

ZF9

Synthetic Zinc Finger

Mol

Doc

k Sc

ore

ZF 5’-Sequence-3’ NO1 GAGGTTGAC 22 TAGGATGGG 13 GGCGCCGAC 04 TAGGCCTAG 05 GTGGAGGCT 26 GACGTAGGA 17 GACGGCGCC 28 TGTGTGGAG 29 GAGGCATGT 2

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

Experimental

Bacterial Two-Hybrid (B2H) Assay

Modified version from Wright et al, 2006

Experimental

B2H Results

Experimental

• Long assay with tedious steps

• Completed with inconclusive results

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

Construction: CPEC

1. Initial PCR adds overlapping regions2. Second PCR attaches the insert to the vector

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

Use CPEC to replace tedious construction steps

Experimental

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 synthesized de novo- FACS analysis and Fluorescence microscopy - Confirm network success

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.

Conclusions

Try something new Apply new ideas Improve ideas

EngineeringHow this fits in:

Custom made synthetic zinc finger repressors

Two plasmid Toggle Switch Controller

Conclusions

Team Members

NCSSM Students

Undergraduate

Peter Fan Aakash Indurkhya

Alyssa Ferris

Kevin Chien

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.

Conclusions