Davidson College SynthAces

1
Davidson College Synth-Aces
Tamar Odle (08), Oscar Hernandez (06), Kristen
DeCelle (06), Andrew Drysdale (07), Matt
Gemberling (06), and Nick Cain (06)
2
Overview of Digital Decoder Project

• Long-term goal detect eight different
  combinations of 3 chemicals
• Use RNA-mediated regulation of protein
  production
• Designed antiswitches, riboswitches, and external
  guide sequences.

3
Visualization of Decoder

• Human-readable output for 8 different
  combinations
• Inputs are detected by aptamers that have on or
  off switches

4
The Building Blocks

• 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

5
A Generic Fluorescent Protein

• Constitutive in some strains
• Repressed in some strains
• Inducible with IPTG
• Strongest degradation tag (LVA)

6
Building Necessary Parts
7
Streamlining Synthetic Biology

• Standardized DNA constructs
• Bio Brick Ends
• Limit the number of preparation steps
• Plug and play process

8
Our Plans

• Modular plasmid construction
• Save intermediate constructs
• Three RNA-mediated controls
• Kinetics of proteins /- LVA

9
Our Intermediate Constructs
Step 2
Step 3
Step 4
10
Inserting RNA Regulator
11
Characterization of Parts in Registry
12
Characterization of I6060
13
Initial Observations
I6060 in
MC4100
DH5a
14
Fluorescence Variations
15
I6060 in 3 Cell Strains
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Fluorescence of I6060 in 3 Cell Strains
July 26, 2005
October 29-30, 2005
Fluorescence/Absorbance
Time (Hours)
17
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.

18
Engineering mRNA to Self-regulate
19
RNA Regulatory Molecules

• RNA regulation provides fast feedback
• Several viable approaches to RNA-mediated
  regulation

20
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

21
General Riboswitch Design

• Lac promoter
• MCT8-4 aptamer
• RBS
• 8 bp spacer

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Forward Riboswitch Construct
ForAptRibo

• Riboswitch ligated to EYFP

23
Alternative Riboswitch Configurations
RevAptRibo
3AptRibo
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Riboswitch Activity
Construct ForAptRibo RevAptRibo
3AptRibo I6060 Exp 1 2
25
Creating Independent RNAs to Regulate Protein
Production
26
Antiswitches

• Adapted to target EYFP (Smolke and Bayer, 2005)
• Regulate yeast protein translation
• Antiswitches tested redesigns not synthesized yet

27
First Generation Antiswitchs

• On antiswitch appears to turn on EYFP

• Off antiswitch also appears to turn on EYFP

• Antiswitch targeting RBS not effective

28
Second Generation Antiswitchs

• Single plasmids
• Shared promoter

29
External Guide Sequences

• Trans-acting RNA
• Off EGS
• On EGS with aptamer?

30
EGS Plasmid
31
Digital Decoder Device Design
32
Digital Decoder Device
Goal Decode combination of three chemicals,
display 0-7 on a digital display
Approach Control display using antiswitches,
responsive to Theophylline Caffeine Malachite
Green
33
Digital Decoder Device
34
Digital Decoder Device
B
0
0
0
C
A
G
0
0
F
D
0
E
35
Digital Decoder Device
B
0
0
0
1
C
A
G
0
0
1
F
D
0
E
36
Digital Decoder Device
B
0
2
0
0
1
2
C
A
G
2
0
0
1
2
F
D
0
2
E
37
Digital Decoder Device
B
0
2
3
0
0
1
2
C
3
A
G
2
3
0
0
1
2
3
F
D
0
2
3
E
38
Digital Decoder Device
B
0
2
3
0
0
1
4
2
C
3
A
4
G
2
3
4
0
0
1
2
3
F
D
4
0
2
3
E
39
Digital Decoder Device
B
0
2
3
5
0
0
1
4
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
40
Digital Decoder Device
B
0
2
3
5
0
0
1
4
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
41
Digital Decoder Device
B
0
2
3
5
7
0
0
1
4
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
42
Digital Decoder Device
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
43
Digital Decoder Device
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
3
6
F
D
0
4
2
5
6
7
0
2
3
5
6
E
44
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
45
Inside E. coli 6
0 0 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
46
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
47
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
48
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
49
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
50
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
51
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
52
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
EYFP coding region
Promoter
Start Codon
53
Inside E. coli 6
0 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
54
Inside E. coli 6
1 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
55
Inside E. coli 6
1 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
56
Inside E. coli 6
1 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
57
Inside E. coli 6
1 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
58
Inside E. coli 6
1 1 0
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
59
Inside E. coli 6
Theophylline Caffeine Malachite Green
1 1 0
Promoter
EYFP coding region
Start Codon
60
Inside E. coli 6
1 1 1
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
61
Inside E. coli 6
1 1 1
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
62
Inside E. coli 6
1 1 1
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
63
Inside E. coli 6
1 1 1
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
64
Inside E. coli 6
1 1 1
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
65
Inside E. coli 6
1 1 1
Theophylline Caffeine Malachite Green
Promoter
EYFP coding region
Start Codon
66
Digital Decoder Device
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
3
6
F
D
0
4
2
5
6
7
0
2
3
5
6
E
67
Thanks to MIT hosts and iGEM organizers
Funding support from HHMI, NIH via MIT, Duke
Endowment, and Davidson College