Synthetic Biology: Design and Characterization of antiswitches in E.coli Matt Gemberling 27 April 2006 Outline Overview of Synthetic Biology Introduce antiswitches Construct antiswitches Results The future of prokaryotic antiswitches Synthetic Biology Caffeine Detector Digital Decoder Device Goal: Given a combination of three chemicals, represent the numbers 0-7 on a digital display Approach: Control fluorescence of display components using antiswitches, responsive to: Theophylline Caffeine Malachite Green Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder Theophyllin e CaffeineMalachite Green 0ABCDEF000 1CD001 2BCDEFG010 3BCGDE011 4AGCD100 5ABGDE101 6ABGDEF110 7BCD111 A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device A F G B C D E E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Multiple strains live in each strip of the decoder Only one strain will fluoresce at any given time, and it will illuminate its entire strip. A particular strain will only fluoresce when its corresponding chemical combination is present Combinations of on and off antiswitches required 7 Two different antiswitches: Off antiswitch: Will suppress expression of YFP in the presence of its ligand On antiswitch: Will allow expression of YFP in the presence of its ligand Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: YFP mRNA Start Codon Theophylline Caffeine Malachite Green UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: YFP coding region Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5 UTR Inside E. coli 6: Start Codon Theophylline Caffeine Malachite Green UTR YFP mRNA Digital Decoder Device A F G B C D E E. coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 RNA Antiswitches Smolke and Bayer (2005) first antiswitches in yeast (S. cerevisiae) On and Off switches were designed RNA-mediated regulation of translation Antiswitch (RNA) Aptamer -Nucleic acid (e.g., RNA) -Binds specific ligand Theophylline Theophylline aptamer Caffeine Ribozymes How do you build antiswitches? Construction of Antiswitches Registry of Standard Biological Parts (http://parts.mit.edu)http://parts.mit.edu Synthesize de novo Registry Offers 2,100 parts Standardized Modular Data available for most parts Characterization Sequences Updated in real-time (wiki) Blue Heron BioBrick prefixes and suffixes Mixed Site Antiswitch Insert Results Verify construction Terminator + YFP Add antiswitch On Off Antiswitch Testing E. coli + antiswitch + theophylline Measure cell density Measure fluorescence Divide fluorescence by cell density Off antiswitch On antiswitch Antiswitch v2.0 Antiswitches were uncontrollable One problem No space between the promoter and first ribozyme E. coli require 5 to 9 base pair space PCR to add these base pairs Results v2.0 Verify construction PCR products Results v2.0 PCR products + Promoter ONOFF Results E. coli + antiswitch + theophylline Time course measurements Conclusions Antiswitches dont function in prokaryotes Pursue alternative mechanisms Future Research Issues with antiswitches Stoicheometry issues Ribozyme cleavage Predicted vs. actual folding of antiswitch RNA stability Rate of initiation Continue with Antiswitches? Future of Synthetic Biology Very appropriate for undergraduates More Ph.D. degrees Imagination and creativity encouraged Malaria medicine Acknowledgements Collaborators O. Hernandez, T. Odle, K. DeCelle, N. Cain, A. Drysdale, M. Cowell, J. Ryan, F. Trappey, Dr. A. M. Campbell, Dr. L. Heyer, Dr. K. Bernd, Dr. D. Wessner, Davidson College Dr. D. Endy and R. Rettberg, MIT T. Bayer, CalTech Financial support from HHMI, MIT, and Davidson Biology Dept. External Guide Sequences