Inexpensive Biosensors based on Cell-Free Extracts

Pitt iGEM 2015Konstantin Borisov, Robert Donahue, Garrett Green, Apurva Patil, Alexander Szul

InspirationPaper-based tests are currently used sparingly:• pH paper• pregnancy tests• glucose meters

The media of paper has huge advantages:

Inexpensive cost of production

Simple storage and transportation

Does not require use of laboratory equipment

Inspiration

The idea:

Could Paper-based Sensors Detect:

Diseases: HIV, Malaria, Salmonella, Cancer

Pollutants: Hormones, Heavy metals, Environmental pharmaceutical pollutants

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

Inspiration

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

• Estrogen:

• Feminization of wildlife

• Drinking water contamination

Environmental Pollutants

• Matrix metalloproteinases:

• Biomarkers for colon, breast, prostate, and intestinal cancers

Cancer Biomarkers

• Model system for future development:

• Anti-MUC1 antibody

• Vascular endothelial growth factor A

Small Protein Detection

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 uninterpretableresults?

Similar concerns of pregnancy tests and knowledge of terminal illness

AnalyteDetection

Transcriptional activation in cell-free extract

Signal Processing

Amplification/Quenching

Signal Detection

Fluorescent proteins/ Colorigenicsubstrates

Investigative Methodology

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. coliBL21 prior to lysis

Creation of Sensor Extract

Culture E. coliBL21 with

desired proteins

Step 1

Lyse by sonication and dialyze

contents

Step 2

Implement extract

contents on biosensor

paper

Step 3

Flash-freeze biosensor for

long term storage

Step 4

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

Estrogen Sensing System

• Based on Carnegie Mellon’s estrogen-sensitive T7 RNA Polymerase

• Can be applied to other analyte-response RNA polymerases

No Estrogen InputEstrogen Input

pT7

X

GFP

pT7

Activated

T7*

GFP

T7*

XGFP

T7*

Activated

T7*T7*

Estrogen Sensing System

No Estrogen InputEstrogen Input

pT7

X

GFP

pT7

Activated

T7*

GFP

T7*

XGFP

T7*

Activated

T7*T7*

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

pProt

E. coli

RNAP

X XGFP

No Protease InputProtease Input

pProt

Protease

pProt GFP

GFPE. coli

RNAP

Protease Sensing System

pProt

E. coli

RNAP

X XGFP

No Protease InputProtease Input

pProt

Protease

pProt GFP

GFPE. coli

RNAP

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

p3H

Activation

Domain

GFP

DBDTP

TP

p3H

Activation

Domain

GFP

GFP

DBDTP

No Input Antibody

X

TP

Antibody Input

Anti-MUC1 antibody detection

Anti-MUC1 antibody detection

Three-Hybrid Versatile System

Amplification

pT3 T3 RNAP

NoT3 RNAP Input

Signal

Amplification

T3 RNAP Input

pT3 GFP

T3

RNAP

GFP

X

pT3 T3 RNAP

pT3 GFP

X

X

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

p3H GFP

DBD

p3H GFP

GFP

DBD

No Input VEGF-A

XVEGF-A

VEGF-A Input

VEGF-A detection

Activation

Domain

Activation

Domain

Proteins in Sensor Extract

Estrogen Sensor Preliminary Results

0

100

200

300

400

500

600

700

800

0 20 40 60 80 100 120

GFP

Flu

ore

scen

ce (

RFU

)

Time (minutes)

ERT7 Extract + pT7, eGFP, 100 uM Estrogen

T7 Extract, No DNA

T7 Extract + pT7, eGFP, 100 nM Estrogen

T7 Extract + pT7, eGFP, 30 uM Estrogen

ERT7 Extract + pT7, eGFP, 10 nM Estrogen

ERT7 Extract + pT7, eGFP, 3 uM Estrogen

T7 Extract + pT7, eGFP, 1 nM Estrogen

T7 Extract + pT7, eGFP, 300 nM Estrogen

T7 Extract + pT7, eGFP, 100 uM Estrogen

ERT7 Extract + pT7, eGFP, 30 nM Estrogen

ERT7 Extract + pT7, eGFP, 10 uM Estrogen

T7 Extract + pT7, eGFP, 3 nM Estrogen

T7 Extract + pT7, eGFP, 1 uM Estrogen

T7 Extract + pT7, eGFP, 300 uM Estrogen

ERT7 Extract + pT7, eGFP, 100 nM Estrogen

ERT7 Extract + pT7, eGFP, 30 uM Estrogen