davidson college synth-aces tamar odle (’08), oscar hernandez (’06), kristen decelle (’06),...
TRANSCRIPT
Davidson College Synth-Aces
Tamar Odle (’08), Oscar Hernandez (’06), Kristen DeCelle (’06), Andrew Drysdale (’07), Matt Gemberling (’06), and Nick Cain (’06)
•Long-term goal: detect eight different
combinations of 3 chemicals
•Use RNA-mediated regulation of protein
production
•Designed antiswitches, riboswitches, and
external guide sequences.
Overview of Digital Decoder Project
• Human-readable output for 8 different combinations
• Inputs are detected by aptamers that have “on” or “off” switches
Visualization of Decoder
• One of our initial goals was to assemble 4 fluorescent proteins:
EYFP, RFP, GFP, and ECFP.
• Measuring kinetics of proteins
• Degradation tags to inhibit the fluorescence of the proteins
• Oddities with I6060 in different strains of E. coli
• Parts still needed
The Building Blocks
A Generic Fluorescent Protein
• Constitutive in some strains
• Repressed in some strains
• Inducible with IPTG
• Strongest degradation tag (LVA)
Streamlining Synthetic Biology
• Standardized DNA constructs
• Bio Brick Ends
• Limit the number of
preparation steps
• “Plug and play” process
Our Plans
• Modular plasmid
construction
• Save intermediate constructs
• Three RNA-mediated controls
• Kinetics of proteins +/- LVA
Fluorescence of I6060 in 3 Cell Strains
Flu
ores
cen
ce/A
bso
rban
ce
Time (Hours)
July 26, 2005 October 29-30, 2005
Why Does Fluorescence Vary?
•Hypothesis 1-Genetic variations between strains.
•Hypothesis 2-Genetic variations between cells.
•Hypothesis 3-Mutation in I6060 plasmid.
Less protein is energetically favorable for cell.
•Hypothesis 4-Switch is flipped turning on proteases.
RNA Regulatory Molecules
• RNA regulation provides fast feedback
• Several viable approaches to RNA-mediated regulation
The Cis-acting Switches
•E. coli contain diverse array of natural riboswitches
•Our design used MTCT8-4 aptamer (Jenison, 1994)
•Previous E. coli riboswitches (Gallivan, 2004)
•Goals:
•minimize aptamer length
•test effects of aptamer position
Antiswitches• Adapted to target EYFP (Smolke and Bayer, 2005)
• Regulate yeast protein translation• Antiswitches tested; redesigns not synthesized yet
First Generation AntiswitchsY Theo On/I6060 Results Compared
0
10000
20000
30000
40000
50000
0 2 4 6 8 10 12 14
Concentration of Theophylline (mM)
Fluorescence/Absorban
ce
7/29/09
9/2/09
9/8/09
Y Theo Off/I6060 Results Compared
0
10000
20000
30000
40000
0 2 4 6 8 10 12 14
Concentration of Theophylline (mM)
Flourescence/Absorban
ce
7/29/09
9/2/09
9/8/09
•“On” antiswitch appears to turn on EYFP
•“Off” antiswitch also appears to turn on EYFP
• Antiswitch targeting RBS not effective
Goal: Decode combination of three chemicals, display 0-7 on a digital display
Approach: Control display using antiswitches, responsive to:
TheophyllineCaffeineMalachite Green
Digital Decoder Device
A
F
G
B
C
D
E
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
0
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
Digital Decoder Device
A
F
G
B
C
D
E
0
0
0
0
0
0
1
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
Digital Decoder Device
A
F
G
B
C
D
E
0
0
0
0
0
0
1
1
2
2
2
2
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
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
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
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
D
E
0
0
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
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
Digital Decoder Device
A
F
G
B
C
D
E
0
0
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
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
Digital Decoder Device
A
F
G
B
C
D
E
0
0
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
5
6
6
6
6
6
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
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
7
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
Digital Decoder Device
Digital Decoder Device
A
F
G
B
C
D
E
0
0
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
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
• 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
A
F
G
B
C
D
E
0
0
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
7
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
Digital Decoder Device
Two different antiswitches:
“Off” antiswitch:
Suppress expression of EYFP in the presence of its ligand
“On” antiswitch:
Allow expression of EYFP in the presence of its ligand
Inside E. coli “6”:0
0
0
Start Codon
Promoter
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
Inside E. coli “6”:
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
YFP coding region
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
EYFP coding region
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
Inside E. coli “6”:
Start Codon
Promoter
0
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Start Codon
Promoter
1
1
0
Theophylline
Caffeine
Malachite Green
Inside E. coli “6”:
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
0
Theophylline
Caffeine
Malachite Green
EYFP coding region
Start Codon
Promoter
Theophylline
Caffeine
Malachite Green
1
1
0
Inside E. coli “6”:
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
1
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
1
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
1
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
1
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
1
Theophylline
Caffeine
Malachite Green
EYFP coding region
Inside E. coli “6”:
Start Codon
Promoter
1
1
1
Theophylline
Caffeine
Malachite Green
EYFP coding region
“Digital Decoder Device”
A
F
G
B
C
D
E
0
0
0
0
0
0
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
7
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