Synthetic Biology Design and Characterization of antiswitches in

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” B E. Coli strain Strips in Decoder Theophyllin e Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCDEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 ABGDEF 1 1 0 7 BCD 1 1 1 C A G F D E

Digital Decoder Device B 0 0 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 0 C A G 0 0 F D 0 E

Digital Decoder Device B 0 0 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 0 1 C A G 0 0 1 F D 0 E

Digital Decoder Device B 0 2 0 0 1 2 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 G 4 AGCD 1 0 0 2 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 A 0 2 C 0 1 F D 0 2 E

Digital Decoder Device B 0 2 3 0 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 0 1 2 3 A C G 2 3 0 2 0 1 3 F D 0 2 3 E

Digital Decoder Device B 0 2 3 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 0 4 0 1 2 3 4 A C G 2 3 4 0 2 0 1 3 4 F 0 2 3 E D

Digital Decoder Device B 0 2 3 5 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 0 4 5 A 0 1 2 3 4 C 0 1 3 4 5 D G 2 3 4 5 0 2 F 0 2 3 5 E

Digital Decoder Device B 0 2 3 5 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 A 0 4 5 6 0 1 2 3 4 C G 6 2 3 4 5 F 0 2 6 0 1 3 4 5 6 6 0 2 3 5 E D

Digital Decoder Device B 0 2 3 5 7 E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 A 0 4 5 6 0 1 2 3 4 7 C G 6 2 3 4 5 F 0 2 6 0 1 3 4 5 6 7 6 0 2 3 5 E D

Digital Decoder Device E. Coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 B 0 2 3 5 7 A 6 0 4 5 6 2 3 4 5 F 0 1 3 4 5 6 7 6 0 2 • A particular strain will only fluoresce when its corresponding chemical combination is present • Combinations of “on” and “off” antiswitches required C G • 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. 0 1 2 3 4 7 6 0 2 3 5 E D

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”: 0 Theophylline 0 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP coding region 5’ UTR Start Codon YFP m. RNA

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 0 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 0 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

Inside E. coli “ 6”: 1 Theophylline 1 Caffeine 1 Malachite Green YFP m. RNA 5’ UTR Start Codon

“Digital Decoder Device” B 7 0 2 3 5 E. coli strain Strips in Decoder Theophylline Caffeine Malachite Green 0 ABCDEF 0 0 0 1 CD 0 0 1 2 BCEFG 0 1 0 3 BCGDE 0 1 1 4 AGCD 1 0 0 5 ABGDE 1 0 1 6 AGDEF 1 1 0 7 BCD 1 1 1 A 0 1 2 3 4 7 6 0 4 5 C G 6 2 3 4 5 F 0 1 3 4 5 6 7 6 0 2 3 5 E D

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 Caffeine Theophylline aptamer

Ribozymes

How do you build antiswitches?

Construction of Antiswitches • Registry of Standard Biological Parts (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

Bio. Brick 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 v 2. 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 v 2. 0 • Verify construction PCR products

Results v 2. 0 PCR products + Promoter ON OFF

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. De. Celle, 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, Cal. Tech • Financial support from HHMI, MIT, and Davidson Biology Dept.

External Guide Sequences