DUKE
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
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
Zinc fingers
Design
Characterization
Experimental
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.
Zinc Fingers
Zinc Finger Arrays
α (or recognition)
helices bind to 3 bp of
DNA with high affinity
Developing assembly methods allow
custom made TFs.
Zinc Fingers
ZFA Assembly Methods
Context-Dependent Assembly
(CoDA)
Pre-screened arrays
Sander et al, 2011
Design
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
Reporter Gene 1
Reporter Gene 2
Design
Network Overview
Controller Plasmid
Characterization
Experimental
Conclusion
Characterization
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-stability
This design accounts for:
• The toggling ability for
the network.
• Easy to determine
network success
• CFP: Blue
• YFP: Yellow
Characterization
Analogous Representation
User inputs and
system outputs are
based on desired
outcome and
response values
Method of
communication
between
remote and TV
stays the same
Characterization
Gene Expression System
No inducers added
Time (minutes)
Characterization
Gene Expression System
Insufficient addition of inducer A (or B)
Time (minutes)
Characterization
Gene Expression System
Sufficient addition of inducer A (or B)
Time (minutes)
Experimental
Conclusion
Experimental
Selection of Zinc Finger Arrays
Screen
Coding
Sequences
Characterization
• BLASTn screen of E. coli genome for ZF
binding site
• Generated by ZiFiT
• Set for Context dependent assembly
• PDB models generated by SWISS-model
and w3DNA
• MolDock algorithm => Free Energy Values
Experimental
Computational Results
MolDock Binding Affinity for Zinc
Finger Transcription Factors
-520
0
1
2
3
4
5
6
7
8
9
ZF1
-530
ZF2
-540
MolDock Score
ZF3
-550
ZF4
ZF5
-560
ZF6
-570
ZF7
-580
ZF8
ZF9
-590
-600
Synthetic Zinc Finger
ZF
1
2
3
4
5
6
7
8
9
5’-Sequence-3’
GAGGTTGAC
TAGGATGGG
GGCGCCGAC
TAGGCCTAG
GTGGAGGCT
GACGTAGGA
GACGGCGCC
TGTGTGGAG
GAGGCATGT
NO
2
1
0
0
2
1
2
2
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
• Long assay with tedious steps
• Completed with inconclusive results
• The construction of B2H reporter strain has
several opportunities for error
Experimental
Construction: CPEC
1. Initial PCR adds overlapping regions
2. 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
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
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
How this fits in:
Engineering
Custom made synthetic
zinc finger repressors
Try something new
Two plasmid Toggle Switch
Controller
Apply new ideas
Improve ideas
Conclusions
Team Members
NCSSM Students
Undergraduate
Peter Fan
Aakash Indurkhya
Kevin Chien
Alyssa Ferris
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.