Fluorogen-Activated Biosensors Yang Choo Eric Pederson Peter Wei - - PowerPoint PPT Presentation

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Fluorogen-Activated Biosensors Yang Choo Eric Pederson Peter Wei - - PowerPoint PPT Presentation

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Promoter Characterization using Fluorogen-Activated Biosensors Yang Choo Eric Pederson Peter Wei Jesse Salazar The Problem Intro. Method Comp. Intro Society Promoter Car Engine Racing Production Environmental Small

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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) Normalized Intensity

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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et)

฀ 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et) ฀ 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