promoter characterization using fluorogen-activated...
TRANSCRIPT
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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
?
?
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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
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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
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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
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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.
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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!
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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.
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Circuit Kit –
Easily Shared and Improved Society Method Intro Comp.
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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
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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
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
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Thank you!
Questions?
visit our Wiki at
http://2012.igem.org/Team:Carnegie_Mellon