Duke iGEM 2014
Methodology Scaling up Synthetic Biology Improving Improving Improving CRISPR Access Education Enabling digital logic with CRISPR/dCas9
CRISPR/Cas9 Background Figure adapted from : Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.
CRISPR/Cas9 Background Figure adapted from : Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.
CRISPR/Cas9 Background Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.
Gene Circuits
Multiple binding sites: Principles Binding of a single dCas9 within the promoter region can almost fully repress expression of a gene. Additional dCas9 bound at the promoter act as redundancies - all must dissociate for expression to occur. Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Bikard D1, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA.
Multiple binding sites: Modeling
Multiple binding sites: Design
Molecular Titration: Principles Molecular titration features pairs of neutralizing molecules. We attempt to recreate the characteristic sigmoidal titration curve on a log-log plot Two approaches to molecular titration: 1. Decoy binding sites to neutralize fully-assembled dCas9 2. Anti-tracrRNA to neutralize tracrRNA Mol Syst Biol. 2009;5:272. doi: 10.1038/msb.2009.30. Epub 2009 May 19.Protein sequestration generates a flexible ultrasensitive response in a genetic network. Buchler NE1, Cross FR.
Decoy binding site: Design Bikard et al.
Decoy binding sites: Modeling *dCas9 becomes the limiting factor for 10 uM decoy
Decoy binding sites: Assembly We have developed a modified PCR method for easily generating large arrays of repeat sequences for insertion into bacteria. Using this scheme, we produced 1X, 6X, and 12X decoy site arrays and inserted them into a high-copy plasmid.
Decoy binding sites: Results 0x 1x 6x 12x Restriction digest of pdCas9, GFP1, and decoy plasmids Decoy arrays remain stable even as we increase the number of repeats. Repeat decoy site arrays are inserted into a high-copy plasmid (~200 copies), producing >2000 repeats per cell!
Decoy binding sites: Results
Decoy binding sites: Next Steps We have a scheme for easily generating large arrays of repeat decoy binding sites (18X, 24X). As we increase the number, we get a sharper and sharper response. We can make decoy sites a better titrant by having dCas9 bind more readily to decoys than real targets. We do so by introducing mismatches into the target binding sites. Finally, we can combine decoy sites with a second titrant...
Anti-tracrRNA titration: Design Bikard et al.
Anti-tracrRNA titration: Modeling Estimated in vivo K d for RNA hybridization is between 1 nM and 100 pM - well within the range for ultrasensitive response
Policy and Practices How can the Duke iGEM team increase access to synthetic biology?
Methodology Scaling up Synthetic Biology Improving Improving Improving CRISPR Access Education 3D Printing of Lab Equipment
Synthetic Biology: Improving Access 3D Printing Lab Equipment with PLA plastic
Why PLA? • Made from lactic acid ⇒ Biodegradable • Yale iGEM 2013 – Synthesize PLA from E. coli – Perform experiments on E. coli using materials produced by E. coli?! • Used by many 3D printers, including the MakerBot
Tube Rack
96-well plate Vortex adaptor
Roller Drum!
Price Comparison Full Product 3D Printed Decrease in Price Purchased Online Equivalent (%) Tube Rack $10-20 $1.62 83.8-91.9% decrease 96-well plate $100 $1.67 98.3% decrease accessory Roller drum $2162 $85.50 96% decrease
Consequently… Buying a 3D printer along with all the materials needed for our final product is cheaper than buying a roller drum!
Future Directions: 3D Printing - Provide access of these designs to the high schools and universities
Future Directions: 3D Printing - Provide access of these designs to the high schools and universities - Develop new designs for lab equipment
Future Directions: 3D Printing - Provide access of these designs to the high schools and universities - Develop new designs for lab equipment - Ultimately, lower the cost of entry into synthetic biology research
Interested? Contact me at matthew.faw@duke.edu for access to .stl files
Methodology Scaling up Synthetic Biology Improving Improving Improving CRISPR Access Education Duke House Course NCSSM iGEM
Synthetic Biology: Improving Education : University Level Duke House Course
House Course? - Student-led, ½ credit course open to all Duke students - Title: Creating Life: the emergence of synthetic biology - Spring 2015 - Taught by Duke iGEM with help and lectures by Duke faculty
Objectives: 1) Provide students with a foundation of understanding upon which they can critically evaluate developments in synthetic biology and society 2) Introduce students to the synthetic biology movement, as well as catalyze a dialogue about the potential applications of the young field and its implications for society
Future Directions: House Course - To transform the House Course into a course that is collaboratively designed and taught by iGEM teams in universities and high schools across the world - Creating MOOCs and a standardized curriculum
Synthetic Biology: Improving Education : High School Level Assisting the NCSSM Team
Objectives 1) Educate high school students on synthetic biology and the iGEM competition 2) Increase public awareness of synthetic biology as bioethical implications of the subject concern everyone
Approach 1: Symposium - Summer Research Symposium - Place for NCSSM students to present research, ideas, and projects to each other and staff of NCSSM - Duke iGEM presented their project and an overview of synthetic biology to feel for interest
Approach 2: NCSSM Seminar - Developed a curriculum to teach synthetic biology and molecular genetics basics to high school students - introducing the idea of an NCSSM iGEM team
Approach 3: Startup of NCSSM iGEM - Creating a project - Teaching wet lab skills - Assisting with beginning stages of project - Registering for HS division
Approach 4: TEDx
Future Ideas: NCSSM iGEM - Spread the curriculum to other iGEM teams at the collegiate level to share with local high schools - Increase interest in high school iGEM - Start public seminars to teach about synthetic biology - similar to the goal of TEDx
Scaling up Synthetic Biology Improving Improving Improving CRISPR Access Education Molecular High School Titration: Decoy Level: NCSSM Binding Sites iGEM University Level: Molecular Duke House Titration: Course anti-tracrRNA 3D Printing Equipment
Acknowledgements Anthony Ciesla 1 , Matthew Farnitano 1 , Matthew Faw 1 , Delta Ghoshal 1 , Garima Tomar 1 , Janan Zhu 1 , Mike Zhu 1 , Charles Cooper 2 , Dr. Nicholas Buchler 346 , Dr. Charles Gersbach 356 1 iGEM team member, 2 iGEM team mentor, 3 Faculty Advisor, 4 Department of Biology, Duke University, Durham, NC, 5 Department of Biomedical Engineering, Duke University, Durham, NC, 6 Duke Center for Genomic and Computational Biology, Duke University, Durham, NC The Pratt School of Engineering, Duke Department of We thank the Lord-Alstadt Foundation for funding the Duke iGEM team. We would also like to thank Dr. Mike Lynch, Chip Bobbert, Biology, and the Duke Center for Genomics and and faculty at the NC School of Science and Mathematics for Computational Biology (GCB) support us, Dr. Buchler, their assistance in this project. and Dr. Gersbach.
Thank you very much for your time. WE WELCOME QUESTIONS!
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