Synthetic Biology: Design and Characterization of antiswitches in E.coli Matt Gemberling 27 April 2006.

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Presentation transcript:

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 ( 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 don’t 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