Inexpensive Biosensors based on Cell-Free Extracts Pitt iGEM 2015 Konstantin Borisov, Robert Donahue, Garrett Green, Apurva Patil, Alexander Szul
Inspiration Paper-based tests are currently used sparingly: • pH paper • pregnancy tests • glucose meters
Inspiration The media of paper has huge advantages: Inexpensive cost of Simple storage and Does not require use of production transportation laboratory equipment
The idea: Could Paper-based Sensors Detect: Diseases: HIV, Malaria, Salmonella, Cancer Pollutants: Hormones, Heavy metals, Environmental pharmaceutical pollutants
Inspiration Pardee et al (2014) successfully used paper-based sensors with freeze-dried cell-free lysates Expressing a significant, Detecting RNAs Dehydrated system for selective signal long term storage Pardee, K., Green, A. A., Ferrante, T., Cameron, D. E., Keyser, A. D., Yin, P., Collins, J. J. "Paper-Based Synthetic Gene Networks" Cell . 159 , 4, 6 November 2014. 940-954.
Pitt iGEM Target Analytes Environmental Pollutants • Estrogen: • Feminization of wildlife • Drinking water contamination Cancer Biomarkers • Matrix metalloproteinases: • Biomarkers for colon, breast, prostate, and intestinal cancers Small Protein Detection • Model system for future development: • Anti-MUC1 antibody • Vascular endothelial growth factor A
Human Practices Questions raised during initial project design If this product How would we would be designed want the final for at-home use Similar concerns product to look how would we of pregnancy tests like? minimize the false and knowledge of positives, false How would the terminal illness negatives, and signal be detected uninterpretable on a paper? results?
Investigative Methodology Transcriptional activation in cell-free extract Analyte Detection Amplification/Quenching Signal Processing Fluorescent proteins/ Colorigenic substrates Signal Detection
Investigative Methodology • Many reporter genes available with different advantages: • GFP – strong signal, readily available • mRFP1 – fluoresces in visible light • LacZ – enzymatic conversion of substrates to colored products • We chose GFP for the majority of our experiments • In future studies, increasing the potential outputs could be quite beneficial
Synthesis of Sensor Extract • Standardized cell-free extract protocol for detection systems • Reduced production costs • Minimized production time; extracts can be made within two days • Proteins of interest can be expressed in E. coli BL21 prior to lysis
Creation of Sensor Extract Step 1 Step 2 Step 3 Step 4 Culture E. coli Lyse by Implement Flash-freeze BL21 with sonication extract biosensor for desired and dialyze contents on long term proteins contents biosensor storage paper
Analyte Detection • Transcriptional activation of specific RNA Polymerases in response to the targeted analyte • Amplification of signal through in vitro transcription and translation • Detectable signal within an hour Cell-free extract Modified estrogen Synthetic repressor Recruitment of RNAP receptive T7 RNA cleaved by a specific to DNA through a 3- polymerase hybrid system protease developed by team CMU Estrogen Protease
Estrogen Sensing System • Based on Carnegie Mellon’s estrogen -sensitive T7 RNA Polymerase • Can be applied to other analyte-response RNA polymerases Estrogen Input No Estrogen Input Activated T7* T7* T7* T7* X X GFP pT7 GFP Activated T7* pT7 GFP
Estrogen Sensing System Estrogen Input No Estrogen Input Activated T7* T7* T7* T7* X X GFP pT7 GFP Activated T7* pT7 GFP
Protease Sensing System • Neither DNA binding domain is strong enough to repress the synthetic promoter by itself, but the presence of both domains in proximity causes repression Protease Input No Protease Input Protease E. coli RNAP X pProt X pProt GFP GFP E. coli RNAP pProt GFP
Protease Sensing System Protease Input No Protease Input Protease E. coli RNAP X pProt X pProt GFP GFP E. coli RNAP pProt GFP
Protease Sensing System
Three-Hybrid Versatile System • Designed novel three-hybrid detection system • Recruits E. coli RNAP to a synthetic promoter through a 3-hybrid contact • Possibilities for contacts are limitless – however they need to be strong
Three-Hybrid Versatile System • Anti-MUC1 antibody sensor utilizes the MUC1 immunogenic epitope as bait • VEGF-A sensor uses a single chain variable fragment as bait • VEGF-A is dimeric with two antibodies capable of binding the protein simultaneous (Chen et al 1999) Chen, Y. et al. "Selection and Analysis of an Optimized Anti-VEGF Antibody: Crystal Structure of an Affinity-matured Fab in Complex with Antigen." J. Mol. Biol. (1999) 293 , 865-881.
Three-Hybrid Versatile System Anti-MUC1 antibody detection Antibody Input No Input Antibody Activation Activation Domain Domain TP TP GFP X TP TP DBD DBD p3H GFP p3H GFP
Three-Hybrid Versatile System Anti-MUC1 antibody detection
Amplification T3 RNAP Input NoT3 RNAP Input Signal Amplification X pT3 T3 RNAP pT3 T3 RNAP X GFP X T3 RNAP pT3 pT3 GFP GFP
Investigative Methodology Optimizing Mechanisms: • DNA decoy hairpins bind RNA polymerases, acting as competitive inhibitors and reducing the amount of active polymerases • Quenching mechanisms minimize leaky expression
Results • Majority of the summer was spent cloning the constructs needed for the four subprojects, as well as optimizing the process of creating sensor extracts
Sensor extract • We created a sensor extract protocol that retains crucial proteins from original cells • These extracts can express genes from plasmids in vitro on paper
Hairpin Quenching Results
Future Project Directions • Pitt iGEM has made sensor extracts for the estrogen and protease projects, and will upload the data to their iGEM page after the Jamboree • Estrogen sensor parts (CMU, BBa_K1732015) and protease sensor parts (Pitt, BBa_K1833008- BBa_K1833010) available in iGEM registry for future teams • Sensor extract protocol available at 2015.igem.org/Team:Pitt/Protocols
Acknowledgements • Dr. Jason Lohmueller, who provided the team with project support and advice, helped bring iGEM to Pitt for the second straight year! • Dr. Alex Deiters, who provided advice at all stages of the project and contributed useful feedback and critique of data collection • Dr. Hanna Salman, who generously provided lab space, and provided supplies and chemicals • Dr. Sanjeev Shroff , who gathered funding from various university sources, and took care of logistics of forming a university team • Dr. Cheryl Telmer from CMU’s iGEM team and Keith Pardee from the Collins Lab at MIT , who provided DNA for our project • The University of Pittsburgh departments that came together to fund the Pitt 2015 iGEM team
Thank you! Questions?
Three-Hybrid Versatile System VEGF-A detection VEGF-A Input No Input VEGF-A Activation Activation Domain Domain VEGF-A GFP X DBD DBD p3H GFP p3H GFP
Proteins in Sensor Extract
Estrogen Sensor Preliminary Results 800 ERT7 Extract + pT7, eGFP, 100 uM Estrogen T7 Extract, No DNA 700 T7 Extract + pT7, eGFP, 100 nM Estrogen T7 Extract + pT7, eGFP, 30 uM Estrogen 600 ERT7 Extract + pT7, eGFP, 10 nM Estrogen GFP Fluorescence (RFU) ERT7 Extract + pT7, eGFP, 3 uM Estrogen 500 T7 Extract + pT7, eGFP, 1 nM Estrogen 400 T7 Extract + pT7, eGFP, 300 nM Estrogen T7 Extract + pT7, eGFP, 100 uM Estrogen 300 ERT7 Extract + pT7, eGFP, 30 nM Estrogen ERT7 Extract + pT7, eGFP, 10 uM Estrogen 200 T7 Extract + pT7, eGFP, 3 nM Estrogen 100 T7 Extract + pT7, eGFP, 1 uM Estrogen T7 Extract + pT7, eGFP, 300 uM Estrogen 0 ERT7 Extract + pT7, eGFP, 100 nM Estrogen 0 20 40 60 80 100 120 Time (minutes) ERT7 Extract + pT7, eGFP, 30 uM Estrogen
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