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.