Promoter Characterization using

Fluorogen-Activated Biosensors

Yang Choo

Eric Pederson

Peter Wei

Jesse Salazar

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Comp.

Unlabeled

black boxes!

• Trial and Error

• Suboptimal Fit

Unlabeled

black boxes?

• Trial and Error

• Suboptimal Fit

Promoter

• Production

• Small constructs

• Single molecule

localization

• Racing

• Environmental

• Small car

Car Engine

The Problem Method Intro Society Intro.

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Traditional methods (qPCR, blotting)

• Invasive – lyse cells

• Labor/Time-Intensive

Current Solutions Comp. Method Society Intro.

Current synthetic biology approach

• Fuse promoters of interest with green

fluorescent proteins

• Indirect measurement of promoter

activities

P

Protein

gfp

RNA

?

?

4

Fluorogen-Activated Biosensor

Advantages

• Translation Efficiency

• Transcription Rate

• Real-time

• Non-invasive

• Modular

Our Solution Comp. Method Society Intro.

Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

Protein

RNA RBS Spinach tRNA

stabilizer FAP

FAP

DNA

Transcription

Translation

5

Spinach and DFHBI Method Comp. Intro Society

Paige et al., Science 2011. http://mfold.rna.albany.edu/?q=mfold

Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

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Ben and MG

http://zhanglab.ccmb.med.umich.edu/I-TASSER/

Method Comp. Intro Society

Szent-Gyorgyi et al., Nature Biotechnology 2007.

Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

Emission spectra of Ben is well-

separated from Spinach

Wavelength (nm) N

orm

aliz

ed In

tens

ity

7

Assumptions for the Model Method Comp. Intro Society

• Spinach and FAP are limiting reactants and will produce signal proportional to

the concentration of the protein or RNA (1:1 ratio)

• Every Spinach and FAP is in the correct conformation to bind to their dye

• Malachite green and DFHBI are both cell permeable

• DFHBI (pKa=5.5) is fully deprotonated at cytosolic pH (6.5-7)

Bacteria

MG

DFHBI

FAP

Spinach

Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

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Dosage Curve -

Spinach

• Dosage curve experiments to determine binding affinities of our constructs in vivo.

Method Comp. Intro Society

KD In vitro

(literature)

KD In vivo

(our results)

437nM ~5µM Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

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• Dosage curve experiments to determine binding affinities of our constructs in vivo.

Method Comp. Intro Society

Dosage Curve -

FAP

KD In vitro

(literature)

KD In vivo

(our results)

320nM ~500nM Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

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T7 Promoters and the

Lac Operator

Traditional inducible promoter

• T7 promoters derive from the T7 bacteriophage and require a specific

RNA polymerase in order to begin transcription

• Lac operator (LacO) binds the LacI repressor, which prevents

transcription. The LacI repressor dissociates when lactose is bound.

IPTG is a lactose analog that is not consumed.

• These promoters are widely used but are not widely represented in the

Registry of Standard Biological Parts. (Only 4 catalogued)

LacO RBS FAP

LacI

T7 RNAP

Method Comp. Intro Society

Spinach T7 Promoter

11

Traditional inducible promoter

• T7 promoters derive from the T7 bacteriophage and require a specific

RNA polymerase in order to begin transcription

• Lac operator (LacO) binds the LacI repressor, which prevents

transcription. The LacI repressor dissociates when lactose is bound.

IPTG is a lactose analog that is not consumed.

• These promoters are widely used but are not widely represented in the

Registry of Standard Biological Parts. (Only 4 catalogued)

LacO

LacI

IPTG

Method Comp. Intro Society

T7 Promoters and the

Lac Operator

RBS FAP Spinach

T7 RNAP

T7 Promoter

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Our BioBricks

Rationally designed T7/lac promoters

• T7 promoters have 3 sections: Recognition site, melting box and the

initiation site.

• We made point mutations to develop mutants that we transformed into

cells analyzed with our biosensors.

• Once we developed a model of the transcription/translation process,

we could determine parameters specific to each promoter.

Recognition Melting

Initiation Lac operator

Method Comp. Intro Society

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Measurements of Real-

Time Fluorescence

1. Our expression strain is BL21(DE3), a strain that contains the gene for T7 RNAP, which we

transformed with a high-copy plasmid (pIVEX).

2. We filled a 96 well plate with 100µL of our transformed cells and added 200µM DFHBI into

half of the wells and 10µM MG into the other half.

3. We added IPTG and took time course measurements for 3.5 hours.

Method Comp. Intro Society

Promoter X

RBS Spinach tRNA

stabilizer FAP END BEG

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Rationally Mutated T7

Promoters

Recognition Melting

Initiation Lac operator

Method Comp. Intro Society

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The Model – Big Picture Comp. Method Intro Society

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The Model - Inputs Comp. Method Intro Society

Promoter

characterization

model

RNA fluorescence

measurements (t, R(t))

Protein fluorescence

measurements (t, P(t))

Outputs

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Promoter characterization model

The Model – Ts & Tl Comp. Method Intro Society

MORE ALGEBRA!!! …

…

Ts [R]

[D] (1 et )

d[R]

dt Ts [D] [R]

Transcriptional strength RNA fluorescence

measurements (t, R(t))

Protein fluorescence

measurements (t, P(t))

d[P]

dt [R] Tl [P]

Translational efficiency

Tl [P] e t

Ts [D]

( ) (1 e t )

Ts [D]

( ) (e t e t )

MORE ALGEBRA!!! …

…

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Promoter characterization model

Ts [R]

[D] (1 et )

Tl [P] e t

Ts [D]

( ) (1 e t )

Ts [D]

( ) (e t e t )

The Model Comp. Method Intro Society

RNA fluorescence

measurements (t, R(t))

Protein fluorescence

measurements (t, P(t))

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Another Option: Code

Modeling Approach Comp. Method Intro Society

RNA fluorescence

measurements (t, R(t))

Protein fluorescence

measurements (t, P(t))

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Human Practices Society Method Intro Comp.

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Sharing and Outreach

Human Practices - Goals

Interactive Relatable Easily shared and

improved

Society Method Intro Comp.

22

Circuit Kit

Promoter X

RBS Spinach

RNA-fluorophore

tRNA

stabilizer FAP END

Malachite

Green Dye

DFHBI Dye

BEG

Electronic analog of our BioBrick design

Dye-Complex

Light Emitting Diode

(LED)

Fluorimeter

Photoresistor

Society Method Intro Comp.

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Circuit Kit - Details Society Method Intro Comp.

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Circuit Kit - Interactive

‘Mini-game’ to find the

best promoter

Society Method Intro Comp.

Winner!

25

Circuit Kit - Relatable

Physical, interactive

Brings experiment/lab to

students

Graphical User Interface plots

realistic graphs

(uses modeling function)

Comprehensive teaching

presentation to introduce

concepts

Society Method Intro Comp.

26

Circuit Kit –

Easily Shared and Improved Society Method Intro Comp.

27

Comp. Intro Society

Submitted

BioBricks Method

Previously… Now!

No characterization

data

Temporal Protein

data

Temporal RNA

data

Leaky RNA levels

Leaky Protein levels

Estimated

Parameters

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What We Accomplished

Devised new system of characterizing promoters

Introduced 3 novel well-characterized T7Lac promoters

Method Intro Comp. Society

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What We Accomplished

Devised new system of characterizing promoters

Introduced 3 novel well-characterized T7Lac promoters

Method Intro Comp. Society

Created a model to analyze the data

30

What We Accomplished

Devised new system of characterizing promoters

Introduced 3 novel well-characterized T7Lac promoters

Method Intro Comp. Society

Created a model to analyze the data

Created a circuit kit to act as a teaching tool

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Future Work

What can be built upon our work

Correlate actual concentration of protein/fluorescence

Characterize more promoters – potential collaborations!

Test the same promoters in different cell strains

Choose other approach for modeling

Method Intro Comp. Society

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Sponsors Method Intro Comp. Society

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Instructors:

• Dr. Cheemeng Tan

• Dr. Natasa Miskov-Zivanov

Advisors:

• Dr. Catalina Achim

• Dr. Diana Marculescu

• Dr. Aaron Mitchell

• Dr. Ge Yang

Acknowledgements Method Intro Comp. Society

Thank you!

Questions?

visit our Wiki at

http://2012.igem.org/Team:Carnegie_Mellon