1. DUKE iGEM
Aakash Indurkhya, Peter Fan, and Alyssa Ferris

2. Designing for the Future
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

3. The Future of Synthetic Biology
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

4. Zinc fingers
Design
Characterization
Experimental

5. 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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6. Zinc Finger Arrays Developing assembly methods allow custom made TFs.
Zinc Fingers
Zinc Finger Arrays
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α (or recognition) helices bind to 3 bp of DNA with high affinity
Developing assembly methods allow custom made TFs.

7. Context-Dependent Assembly (CoDA)
Zinc Fingers
ZFA Assembly Methods
Context-Dependent Assembly (CoDA)
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Pre-screened arrays
Sander et al, 2011

8. Design
Characterization
Experimental
Conclusion

9. The original Genetic Toggle Switch
Design
The original Genetic Toggle Switch
Gardner et al, 2000

10. Characteristics of Toggle Switches
Design
Characteristics of Toggle Switches
Bi-stability
Reporter or marker structural genes
Repressible Constitutive Promoters
Low Basal Transcriptional Noise
Image taken from:

11. Controller Mechanism Split the Toggle Switch into two plasmids:
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

12. Design
Network Overview
Controller Plasmid

13. Characterization
Experimental
Conclusion

14. Graphical Representation
Characterization
Graphical Representation
Multiple repression system serves to activate promoters
This design accounts for:
Reduced transcriptional noise
Activation threshold

15. Graphical Representation
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

16. Graphical Representation
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

17. Method of communication between remote and TV stays the same
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

18. No inducers added Characterization Gene Expression System
Time (minutes)

19. Insufficient addition of inducer A (or B)
Characterization
Insufficient addition of inducer A (or B)
Gene Expression System
Time (minutes)

20. Sufficient addition of inducer A (or B)
Characterization
Sufficient addition of inducer A (or B)
Gene Expression System
Time (minutes)

21. Experimental
Conclusion

22. Selection of Zinc Finger Arrays
Experimental
Selection of Zinc Finger Arrays
BLASTn screen of E. coli genome for ZF binding site
Screen
Generated by ZiFiT
Set for Context dependent assembly
Coding Sequences
PDB models generated by SWISS-model and w3DNA
MolDock algorithm => Free Energy Values
Characterization

23. Computational Results
Experimental
Computational Results ZF
5’-Sequence-3’ NO 1
GAGGTTGAC 2
TAGGATGGG 3
GGCGCCGAC 4
TAGGCCTAG 5
GTGGAGGCT 6
GACGTAGGA 7
GACGGCGCC 8
TGTGTGGAG 9
GAGGCATGT

24. 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

25. Bacterial Two-Hybrid (B2H) Assay
Experimental
Bacterial Two-Hybrid (B2H) Assay
Modified version from Wright et al, 2006

26. B2H Results Long assay with tedious steps
Experimental
B2H Results
Long assay with tedious steps
Completed with inconclusive results
The construction of B2H reporter strain has several opportunities for error

27. Construction: CPEC Initial PCR adds overlapping regions
Experimental
Construction: CPEC
Initial PCR adds overlapping regions
Second PCR attaches the insert to the vector
Use CPEC to replace tedious construction steps

28. 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

29. 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.

30. Engineering How this fits in:
Conclusions
How this fits in:
Engineering
Custom made synthetic zinc finger repressors
Two plasmid Toggle Switch Controller
Try something new
Apply new ideas
Improve ideas

31. Team Members NCSSM Students Undergraduate Conclusions Peter Fan
Aakash Indurkhya
Kevin Chien
Alyssa Ferris

32. 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.