Synthetic Biology: Design and Characterization of antiswitches in E .coli

1
Synthetic Biology Design and Characterization of
antiswitches in E .coli

• Matt Gemberling
• 27 April 2006

2
Outline

• Overview of Synthetic Biology
• Introduce antiswitches
• Construct antiswitches
• Results
• The future of prokaryotic antiswitches

3
Synthetic Biology
4
Caffeine Detector
5
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
6
Digital Decoder Device
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
7
Digital Decoder Device
B
0
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
C
A
G
0
0
F
D
0
E
8
Digital Decoder Device
B
0
0
0
1
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
C
A
G
0
0
1
F
D
0
E
9
Digital Decoder Device
B
0
2
0
0
1
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
2
C
A
G
2
0
0
1
2
F
D
0
2
E
10
Digital Decoder Device
B
0
2
3
0
0
1
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
2
C
3
A
G
2
3
0
0
1
2
3
F
D
0
2
3
E
11
Digital Decoder Device
B
0
2
3
0
0
1
4
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
2
C
3
A
4
G
2
3
4
0
0
1
2
3
F
D
4
0
2
3
E
12
Digital Decoder Device
B
0
2
3
5
0
0
1
4
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
2
5
C
3
A
4
G
2
3
4
5
0
0
1
2
3
F
D
4
5
0
2
3
5
E
13
Digital Decoder Device
B
0
2
3
5
0
0
1
4
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
2
5
C
3
6
A
4
G
2
3
4
5
6
0
0
1
2
3
6
F
D
4
5
6
0
2
3
5
6
E
14
Digital Decoder Device
B
0
2
3
5
7
0
0
1
4
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
2
5
C
3
6
A
4
7
G
2
3
4
5
6
0
0
1
2
3
6
F
D
4
5
6
7
0
2
3
5
6
E
15
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
0
1
2
6
C
0
3
A
4
4
5
7
G
2
3
4
5
6
0
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

3
6
F
D
0
4
2
5
6
7
0
2
3
5
6
E
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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
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Inside E. coli 6
0 0 0
Theophylline Caffeine Malachite Green
YFP mRNA
5 UTR
Start Codon
18
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
19
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
20
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
21
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
22
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
23
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
24
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
25
Inside E. coli 6
Theophylline Caffeine Malachite Green
0 1 0
YFP mRNA
5 UTR
Start Codon
26
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 0
YFP mRNA
5 UTR
Start Codon
27
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 0
YFP mRNA
5 UTR
Start Codon
28
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 0
YFP mRNA
5 UTR
Start Codon
29
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 0
YFP mRNA
5 UTR
Start Codon
30
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 0
YFP mRNA
5 UTR
Start Codon
31
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
32
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
33
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
34
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
35
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
36
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
37
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 1
YFP mRNA
5 UTR
Start Codon
38
Digital Decoder Device
B
0
2
3
5
7
0
1
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
2
6
C
0
3
A
4
4
5
7
G
2
3
4
5
6
0
1
3
6
F
D
0
4
2
5
6
7
0
2
3
5
6
E
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RNA Antiswitches

• Smolke and Bayer (2005) first antiswitches in
  yeast (S. cerevisiae)
• On and Off switches were designed
• RNA-mediated regulation of translation

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Antiswitch (RNA)
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Aptamer

• Nucleic acid (e.g., RNA)
• Binds specific ligand

Caffeine
Theophylline
Theophylline aptamer
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Ribozymes
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How do you build antiswitches?
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Construction of Antiswitches

• Registry of Standard Biological Parts
  (http//parts.mit.edu)

• Synthesize de novo

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Registry

• Offers 2,100 parts
• Standardized
• Modular
• Data available for most parts
• Characterization
• Sequences
• Updated in real-time (wiki)

47
Blue Heron
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BioBrick prefixes and suffixes
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Mixed Site
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Antiswitch Insert
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Results

• Verify construction

Terminator YFP
Add antiswitch
On
Off
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Antiswitch Testing

• E. coli antiswitch theophylline
• Measure cell density
• Measure fluorescence
• Divide fluorescence by cell density

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Off antiswitch
On antiswitch
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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

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Results v2.0

• Verify construction

PCR products
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Results v2.0
PCR products Promoter
ON
OFF
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Results

• E. coli antiswitch theophylline
• Time course measurements

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Conclusions

• Antiswitches dont function in prokaryotes
• Pursue alternative mechanisms

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Future Research

• Issues with antiswitches
• Stoicheometry issues
• Ribozyme cleavage
• Predicted vs. actual folding of antiswitch
• RNA stability
• Rate of initiation

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Continue with Antiswitches?
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Future of Synthetic Biology

• Very appropriate for undergraduates
• More Ph.D. degrees
• Imagination and creativity encouraged
• Malaria medicine

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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.

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External Guide Sequences
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