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DUKE iGEM
Aakash Indurkhya, Peter Fan, and Alyssa Ferris
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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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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
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Zinc fingers
Design
Characterization
Experimental
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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
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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.
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ZFA Assembly MethodsContext-Dependent Assembly
(CoDA)
Pre-screened arrays Sander et al, 2011
Zinc Fingers
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Characterization
Experimental
Conclusion
Design
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The original Genetic Toggle Switch
Gardner et al, 2000
Design
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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
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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
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Network Overview
Controller Plasmid
Design
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Characterization
Experimental
Conclusion
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Graphical Representation
• Multiple repression system serves to activate promoters
This design accounts for:• Reduced transcriptional noise• Activation threshold
Characterization
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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
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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
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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
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No inducers added
Time (minutes)
Gen
e Ex
pres
sion
Sys
tem
Characterization
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Insufficient addition of inducer A (or B)G
ene
Expr
essi
on S
yste
m
Time (minutes)
Characterization
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Sufficient addition of inducer A (or B)G
ene
Expr
essi
on S
yste
m
Time (minutes)
Characterization
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Experimental
Conclusion
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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
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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
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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
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Bacterial Two-Hybrid (B2H) Assay
Modified version from Wright et al, 2006
Experimental
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B2H Results
Experimental
• Long assay with tedious steps
• Completed with inconclusive results
• The construction of B2H reporter strain has several opportunities for error
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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
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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 synthesized de novo- FACS analysis and Fluorescence microscopy - Confirm network success
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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
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Try something new Apply new ideas Improve ideas
EngineeringHow this fits in:
Custom made synthetic zinc finger repressors
Two plasmid Toggle Switch Controller
Conclusions
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Team Members
NCSSM Students
Undergraduate
Peter Fan Aakash Indurkhya
Alyssa Ferris
Kevin Chien
Conclusions
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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