Inexpensive Biosensors based on Cell-Free Extracts

Transcription

1 Inexpensive Biosensors based on Cell-Free Extracts Pitt igem 2015 Konstantin Borisov, Robert Donahue, Garrett Green, Apurva Patil, Alexander Szul

2 Inspiration Paper-based tests are currently used sparingly: ph paper pregnancy tests glucose meters

3 Inspiration The media of paper has huge advantages: Inexpensive cost of production Simple storage and transportation Does not require use of laboratory equipment

4 The idea: Could Paper-based Sensors Detect: Diseases: HIV, Malaria, Salmonella, Cancer Pollutants: Hormones, Heavy metals, Environmental pharmaceutical pollutants

5 Inspiration Pardee et al (2014) successfully used paper-based sensors with freeze-dried cell-free lysates Expressing a significant, selective signal Detecting RNAs Dehydrated system for 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

6 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

7 Human Practices Questions raised during initial project design How would we want the final product to look like? How would the signal be detected on a paper? If this product would be designed for at-home use how would we minimize the false positives, false negatives, and uninterpretable results? Similar concerns of pregnancy tests and knowledge of terminal illness

8 Investigative Methodology Analyte Detection Transcriptional activation in cell-free extract Signal Processing Signal Detection Amplification/Quenching Fluorescent proteins/ Colorigenic substrates

9 Investigative Methodology Many reporter genes available with different advantages: strong signal, readily available mrfp1 fluoresces in visible light LacZ enzymatic conversion of substrates to colored products We chose for the majority of our experiments In future studies, increasing the potential outputs could be quite beneficial

10 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

11 Creation of Sensor Extract Step 1 Step 2 Step 3 Step 4 Culture E. coli BL21 with desired proteins Lyse by sonication and dialyze contents Implement extract contents on biosensor paper Flash-freeze biosensor for long term storage

12 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 receptive T7 RNA polymerase developed by team CMU Estrogen Synthetic repressor cleaved by a specific protease Protease Recruitment of RNAP to DNA through a 3- hybrid system

13 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 T7* T7* Activated T7* Activated T7* T7* X pt7 X pt7

14 Estrogen Sensing System Estrogen Input No Estrogen Input T7* T7* Activated T7* Activated T7* T7* X pt7 X pt7

15 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 Protease No Protease Input pprot E. coli RNAP X pprot X E. coli RNAP pprot

16 Protease Sensing System Protease Input Protease No Protease Input pprot E. coli RNAP X pprot X E. coli RNAP pprot

17 Protease Sensing System

18 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

19 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,

20 Three-Hybrid Versatile System Anti-MUC1 antibody detection Antibody Input No Input Antibody TP Activation Domain Activation Domain TP TP DBD TP DBD X p3h p3h

21 Three-Hybrid Versatile System Anti-MUC1 antibody detection

22 Amplification T3 RNAP Input NoT3 RNAP Input Signal Amplification X pt3 T3 RNAP T3 RNAP pt3 X T3 RNAP X pt3 pt3

23 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

24 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

25 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

26 Hairpin Quenching Results

27 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_K ) and protease sensor parts (Pitt, BBa_K BBa_K ) available in igem registry for future teams Sensor extract protocol available at 2015.igem.org/Team:Pitt/Protocols

28 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

29 Thank you! Questions?

30 Three-Hybrid Versatile System VEGF-A detection VEGF-A Input No Input VEGF-A Activation Domain Activation Domain VEGF-A DBD DBD X p3h p3h

31 Proteins in Sensor Extract

32 Fluorescence (RFU) Estrogen Sensor Preliminary Results ERT7 Extract + pt7, e, 100 um Estrogen T7 Extract, No DNA T7 Extract + pt7, e, 100 nm Estrogen T7 Extract + pt7, e, 30 um Estrogen ERT7 Extract + pt7, e, 10 nm Estrogen 500 ERT7 Extract + pt7, e, 3 um Estrogen T7 Extract + pt7, e, 1 nm Estrogen T7 Extract + pt7, e, 300 nm Estrogen T7 Extract + pt7, e, 100 um Estrogen ERT7 Extract + pt7, e, 30 nm Estrogen ERT7 Extract + pt7, e, 10 um Estrogen T7 Extract + pt7, e, 3 nm Estrogen 100 T7 Extract + pt7, e, 1 um Estrogen Time (minutes) T7 Extract + pt7, e, 300 um Estrogen ERT7 Extract + pt7, e, 100 nm Estrogen ERT7 Extract + pt7, e, 30 um Estrogen