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DUKE iGEM

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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DUKE iGEM

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  1. DUKEiGEM Aakash Indurkhya, Peter Fan, and Alyssa Ferris

  2. 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

  3. 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

  4. Zinc fingers Design Characterization Experimental

  5. 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.

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

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

  8. Design Characterization Experimental Conclusion

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

  10. 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

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

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

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

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

  21. Experimental Conclusion

  22. Experimental Selection of Zinc Finger Arrays

  23. Experimental Computational Results

  24. 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

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

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

  27. 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

  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 synthesizedde 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. 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

  31. Conclusions Team Members NCSSM Students Undergraduate Aakash Indurkhya Peter Fan 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.

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