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)

Building Necessary Parts

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

Our Intermediate Constructs

Step 2Step 3

Step 4

Inserting RNA Regulator

Characterization of Parts in Registry

Part AFl

uore

scen

ce

Time

Part B

Fluo

resc

ence

Time

Characterization of I6060

Initial Observations

MC4100 DH5I6060 in

Fluorescence Variations

I6060 in

DH5 (June 23)

I6060 in

DH5 (June 24)

I6060 in

MC4100 (June 26)

I6060 in 3 Cell Strains

MC

4100

DH

5

JM109

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.

Engineering mRNA to Self-regulate

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

General Riboswitch Design

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

Forward Riboswitch Construct

Riboswitch ligated to EYFP

ForAptRibo

Alternative Riboswitch Configurations

3AptRibo RevAptRibo

Riboswitch Activity

Construct

ForAptRibo

RevAptRibo

3AptRibo

I6060

Exp #

1

2

Creating Independent RNAs to Regulate Protein

Production

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

Second Generation Antiswitchs

•Single plasmids

•Shared promoter

External Guide Sequences

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

EGS Plasmid

Digital Decoder Device Design

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

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3

3

3

3

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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

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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

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4

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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

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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

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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

Thanks to MIT hosts and iGEM organizers

Funding support from: HHMI, NIH via MIT, Duke Endowment, and Davidson College