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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.
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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
“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
B C A G F D E “Digital Decoder Device”
Digital Decoder Device B 0 0 0 C A G 0 0 F D 0 E
Digital Decoder Device B 0 0 0 1 C A G 0 0 1 F D 0 E
Digital Decoder Device B 0 2 0 0 1 2 C A G 2 0 0 2 1 F D 0 2 E
Digital Decoder Device B 0 2 3 0 0 1 2 C 3 A G 2 3 0 0 2 1 3 F D 0 2 3 E
Digital Decoder Device B 0 2 3 0 0 4 1 2 C 3 A 4 G 2 3 4 0 0 2 1 3 F 4 D 0 2 3 E
Digital Decoder Device B 0 2 3 5 0 0 4 1 5 2 C 3 A 4 G 2 3 4 5 0 0 2 1 3 F 4 D 5 0 2 3 5 E
Digital Decoder Device B 0 2 3 5 0 0 4 1 5 2 C 6 3 A 4 G 6 2 3 4 5 0 0 2 1 6 3 F 4 D 5 6 6 0 2 3 5 E
Digital Decoder Device B 0 2 3 5 7 0 0 4 1 5 2 C 6 3 A 4 7 G 6 2 3 4 5 0 0 2 1 6 3 F 4 D 5 6 7 6 0 2 3 5 E
Digital Decoder Device B 0 2 3 5 7 0 1 6 2 C 0 3 A 4 4 5 7 G 6 2 3 4 5 0 • 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 1 6 3 F 0 4 D 2 5 6 7 6 0 2 3 5 E
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 0 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E. coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
YFP coding region Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E. coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E. coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon
“Digital Decoder Device” B 7 0 2 3 5 0 1 6 2 C 0 3 A 4 4 5 7 G 6 2 3 4 5 0 1 6 3 F 0 4 D 2 5 6 7 6 0 2 3 5 E
RNA Antiswitches • Smolke and Bayer (2005) first antiswitches in yeast (S. cerevisiae) • “On” and “Off” switches were designed • RNA-mediated regulation of translation
Aptamer • Nucleic acid (e.g., RNA) • Binds specific ligand Caffeine Theophylline Theophylline aptamer
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)