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

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decaf regular espresso

Relative expression

GFP

YFP

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:

TheophyllineCaffeineMalachite Green

“Digital Decoder Device”

A

F

G

B

C

D

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E. Coli strain

Strips in Decoder

Theophylline

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

A

F

G

B

C

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0

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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

Digital Decoder Device

A

F

G

B

C

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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

Digital Decoder Device

A

F

G

B

C

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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

Digital Decoder Device

A

F

G

B

C

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E

0

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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

Digital Decoder Device

A

F

G

B

C

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E

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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

Digital Decoder Device

A

F

G

B

C

D

E

0

0

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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

Digital Decoder Device

A

F

G

B

C

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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

Digital Decoder Device

A

F

G

B

C

D

E

0

0

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0

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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

Digital Decoder Device

Digital Decoder Device

A

F

G

B

C

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E

0

0

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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

• 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

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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

Start Codon

Theophylline

Caffeine

Malachite Green

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

YFP mRNA

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

YFP coding region

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

0

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

0

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

YFP mRNA 5’ UTR

Inside E. coli “6”:

Start Codon

Theophylline

Caffeine

Malachite Green

1

1

1

5’ UTR YFP mRNA

“Digital Decoder Device”

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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

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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Ribozymes

How do you build antiswitches?

Construction of Antiswitches

• Registry of Standard

Biological Parts (

http://parts.mit.edu)

• Synthesize de novo

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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 constructionTerminator + 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 constructionPCR products

Results v2.0

PCR products + Promoter

ON OFF

Results

• E. coli + antiswitch + theophylline

• Time course measurements

Theoretical Antiswitch Results

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Theophylline Conc. (mmol)

Fluor/abs

On Off

Hour One

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0 mmol 0.1 mmol 1 mmol 5 mmol

Theo Conc. in mmol

Fluor/ abs.

Hour Two

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0 mmol 0.1 mmol 1 mmol 5 mmol

Theo Conc in mmol

Fluor/abs.

Hour Three

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0 mmol 0.1 mmol 1 mmol 5 mmol

Theo Conc in mmol

Fluor/ Abs.

Hour Four

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Theo conc. in mmol

Fluor/ abs.

Hour Five

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Theo Conc. in mmol

Fluor/abs.

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