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 constructs • Small car • Single molecule localization Unlabeled Unlabeled black boxes! black boxes? • Trial and Error • Trial and Error • Suboptimal Fit • Suboptimal Fit 2
Current Solutions Method Intro. Comp. Society Traditional methods (qPCR, blotting) • Invasive – lyse cells • Labor/Time-Intensive gfp P ? Current synthetic biology approach • Fuse promoters of interest with green RNA fluorescent proteins ? Indirect measurement of promoter • activities Protein 3
Our Solution Method Intro. Comp. Society Promoter X tRNA DNA END BEG RBS FAP Spinach stabilizer Fluorogen-Activated Biosensor Transcription Advantages • Translation Efficiency tRNA Spinach RBS FAP RNA stabilizer • Transcription Rate • Real-time • Non-invasive Translation • Modular Protein FAP 4
Spinach and DFHBI Method Intro Society Comp. Promoter X Paige et al., Science 2011. http://mfold.rna.albany.edu/?q=mfold 5 tRNA END BEG FAP Spinach RBS stabilizer
Ben and MG Method Intro Society Comp. Emission spectra of Ben is well- separated from Spinach Normalized Intensity Wavelength (nm) Promoter X http://zhanglab.ccmb.med.umich.edu/I-TASSER/ 6 Szent-Gyorgyi et al., Nature Biotechnology 2007. tRNA END BEG FAP Spinach RBS stabilizer
Assumptions for the Model Method Intro Society Comp. FAP Bacteria MG DFHBI Spinach • 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 (pK a =5.5) is fully deprotonated at cytosolic pH (6.5-7) Promoter X 7 tRNA END BEG FAP Spinach RBS stabilizer
Dosage Curve - Spinach Method Intro Society Comp. • Dosage curve experiments to determine binding affinities of our constructs in vivo . K D In vitro K D In vivo (literature) (our results) 437nM ~5µM Promoter X 8 tRNA END BEG FAP Spinach RBS stabilizer
Dosage Curve - FAP Method Intro Society Comp. • Dosage curve experiments to determine binding affinities of our constructs in vivo . K D In vitro K D In vivo (literature) (our results) Promoter X 320nM ~500nM 9 tRNA END BEG FAP Spinach RBS stabilizer
T7 Promoters and the Lac Operator Method Intro Society Comp. 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) T7 RNAP LacI Spinach FAP LacO RBS T7 Promoter 10
T7 Promoters and the Lac Operator Method Intro Society Comp. 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) T7 RNAP IPTG LacI Spinach FAP LacO RBS T7 Promoter 11
Our BioBricks Method Intro Society Comp. 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. Melting Lac operator Recognition Initiation 12
Measurements of Real- Time Fluorescence Method Intro Society Comp. 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. Promoter X 13 tRNA END BEG FAP Spinach RBS stabilizer
Rationally Mutated T7 Promoters Method Intro Society Comp. Melting Lac operator Recognition Initiation 14
The Model – Big Picture Method Comp. Society Intro 15
The Model - Inputs Method Comp. Society Intro RNA fluorescence measurements ( t , R ( t )) Promoter Outputs characterization Protein fluorescence model measurements ( t , P ( t )) 16
The Model – Ts & Tl Method Comp. Society Intro Transcriptional strength RNA fluorescence d [ R ] measurements ( t , R ( t )) Ts [ D ] [ R ] dt … MORE ALGEBRA!!! … [ R ] Ts [ D ] (1 e t ) Promoter characterization model Translational efficiency Protein fluorescence d [ P ] [ R ] Tl [ P ] measurements ( t , P ( t )) dt … MORE ALGEBRA!!! … [ P ] e t Tl Ts [ D ] Ts [ D ] ( ) ( e t e t ) ( ) (1 e t ) 17
The Model Method Comp. Society Intro Promoter characterization model RNA fluorescence measurements ( t , R ( t )) Protein fluorescence measurements ( t , P ( t )) [ R ] Ts [ D ] (1 e t ) [ P ] e t Tl Ts [ D ] Ts [ D ] ( ) ( e t e t ) ( ) (1 e t ) 18
Modeling Approach Method Comp. Society Intro Another Option: Code RNA fluorescence Protein fluorescence measurements ( t , P ( t )) measurements ( t , R ( t )) 19
Human Practices Society Method Intro Comp. 20
Human Practices - Goals Society Method Intro Comp. Sharing and Outreach Easily shared and Interactive Relatable improved 21
Circuit Kit Society Method Intro Comp. Electronic analog of our BioBrick design Dye-Complex Fluorimeter DFHBI Dye Light Emitting Diode Malachite Photoresistor (LED) Green Dye Promoter X Spinach tRNA END BEG FAP RBS stabilizer RNA-fluorophore 22
Circuit Kit - Details Society Method Intro Comp. 23
Circuit Kit - Interactive Society Method Intro Comp. ‘ Mini-game ’ to find the best promoter Winner! 24
Circuit Kit - Relatable Society Method Intro Comp. Physical, interactive Brings experiment/lab to students Graphical User Interface plots realistic graphs (uses modeling function) Comprehensive teaching presentation to introduce concepts 25
Circuit Kit – Easily Shared and Improved Society Method Intro Comp. 26
Submitted BioBricks Method Intro Society Comp. Temporal Protein data Temporal RNA data Leaky RNA levels No characterization Leaky Protein levels data Estimated Parameters Previously… Now! 27
What We Accomplished Method Society Intro Comp. Devised new system of characterizing promoters Introduced 3 novel well-characterized T7Lac promoters 28
What We Accomplished Method Society Intro Comp. Devised new system of characterizing promoters Introduced 3 novel well-characterized T7Lac promoters Created a model to analyze the data 29
What We Accomplished Method Society Intro Comp. Devised new system of characterizing promoters Introduced 3 novel well-characterized T7Lac promoters Created a model to analyze the data Created a circuit kit to act as a teaching tool 30
Future Work Method Society Intro Comp. 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 31
Sponsors Method Society Intro Comp. 32
Acknowledgements Method Society Intro Comp. Instructors: • Dr. Cheemeng Tan • Dr. Natasa Miskov-Zivanov Advisors: • Dr. Catalina Achim • Dr. Diana Marculescu • Dr. Aaron Mitchell • Dr. Ge Yang 33
Thank you! Questions? visit our Wiki at http://2012.igem.org/Team:Carnegie_Mellon
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