DUKE iGEM Aakash Indurkhya, Peter Fan, and Alyssa Ferris

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

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

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. SCRIPT NEEDED

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

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

Design Characterization Experimental Conclusion

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

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: http://parts.mit.edu/igem07/index.php/Tokyo/sunaba2

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

Design Network Overview Controller Plasmid

Characterization Experimental Conclusion

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

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

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

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

No inducers added Characterization Gene Expression System Time (minutes)

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

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

Experimental Conclusion

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

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

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

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

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

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 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.

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

Team Members NCSSM Students Undergraduate Conclusions 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.