Hello all! We’ve tried to minimize the overlap but several slides are best viewed animated! - MIT iGEM RNA Strand Displacement for Sensing, Information Processing, and Actuation in Mammalian Cells America’s East Regional 10.13.2012 1
Brief Overview of Nucleic Acid Technology DNA Structure DNA Computing Signal Amplification Static AIM: Develop strand displacement as a mechanism to build in vivo Zhang et al ., Science 2007 Rothemund, Nature 2006 synthetic circuits Dynamic Digital Logic A AND B AND (C OR D) AND (E OR F) A A Omabegho et al. , Science 2009 Seelig et al, Science 2006
Attractiveness of Nucleic Acid Computation 40 Number of Promoters / dsGates Trancription-translational circuits 35 Strand displacement circuits 30 25 Qian et al., Science 2011 20 15 10 5 Moon et al., Nature 2012 0 2000 2002 2004 2006 2008 2010 2012 Year Analysis of publications of the Winfree group Purnick et al., Nature MCB 2009 3
Increased Complexity / Smaller Footprint Moon et al., Nature 2012 Transcriptional-translational circuit: ~13000 bp Strand displacement circuit: 100 bp 4 Much smaller nucleotide footprint!
Increased Complexity / Smaller Footprint Large Sophisticated Circuits Enabled By: • Decreased Size • Simple Combinatorial Design Space • Ease of Composition • Tunability 5 Much smaller nucleotide footprint!
Implementation Strategies T long S long T long * S long * In vitro In vivo Utilize RNA Mammalian Cells 1 cell cycle vs. 24 hours 72 cell cycles Nucleic Acid Computing Strategy Toehold Hybridization domain
Toehold-Mediated Strand Displacement Input Strand Output Strand Output Strand Input Strand Gate 7
Deliver RNA Demonstrate In Vitro Design & Test NOT Gate RNA Strand Displacement Demonstrate In Vivo RNA Strand Displacement Design & Test mRNA Sensor Produce Short RNAs In Vivo
In Vitro Strand Displacement Using RNA S6 T S6 S6 S1 T S6 + + T* S6* T* S6* Incorrect Input Input Strand Reporter Fluorescent Complex Waste Strand 9 Data collected by Eerik
Processing Using Strand Displacement Qian et al. 2011 Nothing compatible ? AND OR NOT True if both inputs true True if at least one input is true Inverts a signal 10
Inversion Using Strand Displacement Design: Buffer (C) Fuel / Catalyst (D) Dynamic Gate (A) Input Strand Downstream Input (B) Operation: Low Input Dynamic Gate (A) Downstream Downstream Input (B) Input (B) B is free to act downstream! C is displaced.
Inversion Using Strand Displacement Design: Buffer (C) Fuel / Catalyst (D) Dynamic Gate (A) Input Strand Downstream Input (B) Operation: High Input Dynamic Gate (A) Input Strand Downstream Input (B) B is trapped, cannot act downstream! C is ‘stable’
Our NOT Gate In Vitro Design Simulate Test x5
Deliver RNA Demonstrated In Vitro Designed & Tested NOT Gate RNA Strand Displacement Demonstrated In Vivo RNA Strand Displacement Designed & Tested mRNA Sensor Produced Short RNAs In Vivo
In Vivo RNA Delivery T = 0h T = 2h T = 3h T = 4h HEK293 cell Vesicle Tagged RNA Video Here Experiment by Katie
Delivered RNA Demonstrated In Vitro Designed & Tested NOT Gate RNA Strand Displacement Demonstrate In Vivo RNA Strand Displacement Designed & Tested mRNA Sensor Produced Short RNAs In Vivo
Design Test 17
In Vivo Strand Displacement: Iteration 1 S T S S T S + + T * S* T* S* Input Strand Reporter Fluorescent Complex Waste 18 Transfection by Katie, FACS by Nathan
In Vivo Strand Displacement: Iteration 2 S long T long S long S long T long S long + + T long * S long * T long * S long * Input Strand Reporter Fluorescent Complex Waste ~6 fold increase in red 19 Nucleofection by Giulio, FACS by Rob
In Vivo Strand Displacement Summary Delivered RNA Demonstrated In Vitro Designed & Tested NOT Gate RNA Strand Displacement Demonstrated In Sensors Vivo RNA Strand Displacement Short RNA Transcription
Sensing mRNA for Cellular Interfacing Input mRNA + Design objectives and Sensor constraints: • orthogonality Fuel • three-letter code • accessibility + Downstream Short RNA Input Output 21
Sensing mRNA for Cellular Interfacing Software Rendered eBFP2 mRNA 22 Design objectives and constraints: orthogonality, three-letter code, accessibility
Sensing mRNA In Vitro Using DNA 2.03 2.62 2.28 3.18 mRNA + Sensor + Reporter = Fluorescence 1 2 3 4 23 Data by Eerik and Chelsea
Delivered RNA Demonstrated In Vitro Designed & Tested NOT Gate RNA Strand Displacement Demonstrated In Vivo RNA Strand Displacement Design & Test mRNA Sensor Produce Short RNAs In Vivo
Producing Short RNAs In Vivo 67bp hairpin U6 TetO FF1 Hef1A eYFP-4xFF1 Hef1A TagBFP Transfection marker Knockdown of Hef1A:eYFP-4xFF1 using U6-TetO:FF1 25 Transfection by Linh, FACS by Nathan
Summary of Achievements Delivered RNA Demonstrated In Vitro Designed & Tested NOT Gate RNA Strand Displacement Demonstrated In Vivo RNA Strand Displacement Produce Short RNAs Design & Test In Vivo mRNA Sensor
New MammoBlocks / BioBricks 37 Best 22 Parts Submitted To Registry Biobricks 40 10 Regulatory 3 Promoters MammoBlocks BBa_K779200 Composite Parts BBa_K779201 BBa_K779400 BBa_K779405 BBa_K779202 BBa_K779401 BBa_K779406 37 Logic Parts for Strand 4 Hammerhead Ribozyme BBa_K779402 BBa_K779407 Displacement BBa_K779403 BBa_K779408 Coding Sequences BBa_K779404 BBa_K779409 BBa_K779500 BBa_K779501 BBa_K779502 BBa_K779315 2 Transcriptional BBa_K779503 BBa_K779504 BBa_K779100 BBa_K779316 Regulators BBa_K779101 BBa_K779102 BBa_K779103 BBa_K779317 BBa_K779104 BBa_K779105 BBa_K779106 Bba_K779318 BBa_K779305 BBa_K779107 BBa_K779108 BBa_K779109 BBa_K779308 13 Reporters BBa_K779110 BBa_K779111 BBa_K779112 BBa_K779113 BBa_K779114 BBa_K779115 BBa_K779300 BBa_K779307 10 Generators BBa_K779116 BBa_K779117 BBa_K779118 BBa_K779301 BBa_K779309 BBa_K779119 BBa_K779120 BBa_K779121 BBa_K779600 BBa_K779601 BBa_K779302 BBa_K779310 BBa_K779122 BBa_K779123 BBa_K779124 BBa_K779602 BBa_K779603 BBa_K779303 BBa_K779311 BBa_K779125 BBa_K779126 BBa_K779127 BBa_K779604 BBa_K779605 BBa_K779304 BBa_K779312 BBa_K779128 BBa_K779129 BBa_K779130 BBa_K779306 BBa-K779313 BBa_K779606 BBa_K779607 BBa_K779131 BBa_K779314 BBa_K779608 BBa_K779609
Long Term Impact on Society In Vivo RNA Strand Displacement Sophisticated Circuits in Mammalian Cells Control Circuitry For Smart Immune Cancer Mammalian Protein System Detection Production Organ Regeneration
Outreach: Middle School, High School, College Summer HSSP: Educating local middle school students Biology Lecture Series: Synthetic Biology , organized and taught by iGEM students Educate the public about applications, practices, and opportunities in synthetic biology Splash: Educating local high school students MIT Educational Studies Program Coming soon! November 17, 2012 Inform soon-to-be college students about a potential field Wellesley: Building multi-university communities Use case for Wellesley HCI software Bridging gap between tool designer and end-user MIT: IAP Synthetic Biology Class “Engineer Your Own Bacteria” 2 week lecture, wet lab Fundraised, Organized, Taught by MIT iGEMers 29
Acknowledgments MIT iGEM 2012 Team Keren Greenbaum, Giulio Alighieri, Divya Israni, Lealia Xiong, Jenna Klein, Katie Bodner, Nathan Kipniss, Felix Sun, Ala’a Siam, Kristjan Eerik Kaseniit, Robert Learsch, Linh Vuong, Chelsea Voss, Wilson Louie, Jonathan Elzur, Eta Atolia Coordinators: Additional thanks to: Lulu Qian Ron Weiss (faculty) Peter Andrew Carr Jonathan Babb Nevin M. Summers Deepak Mishra Timothy Lu Domatilla Del Vecchio Alice M. Rushforth Lab Shift Monitors: Roger Kamm Narendra Maheshri Jameel Zayed Natalie Kuldell Kenneth H. Hu Christopher Voigt Leanna S. Morishini Feng Zhang Mariya Barch Jacquin Niles Mark Andrew Kristala L. Jones Keibler Prather Nathan S. Rahul Sarpeshkar Lachenmyer BU-Wellesley Sebastien Lemire iGEM Team Thanks to our sponsors for their generous support!
Thank you! Questions?
Full NOT Gate Reaction Diagram Case: No input present high output signal Reversible Dynamic Gate (A) No Input Downstream Strand Input (B) Downstream Input (B) reacts: Downstream Signal! Buffer (C) Input (B) Fuel / Catalyst (D) Case: Input present low output signal Trapped! Dynamic Gate (A) Irreversible Input Strand B 33
AND and OR Logic 34 Qian et al. 2011
Strand Displacement Reactions 35 Qian et al. 2011
Optimizing Transfection of 2’ -O-Me dsRNA 36
In Vitro Strand Displacement: Iteration 2 S long/bulge T long S long S long/bulge T long S long + + T long * S long * T long * S long * Input Strand Reporter Fluorescent Complex Waste 37
In Vivo Strand Displacement: Iteration 2 S long T long S long S long T long S long + + T long * S long * T long * S long * Input Strand Reporter Fluorescent Complex Waste 38
Inducible Expression Systems
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