on Cell-Free Extracts Pitt iGEM 2015 Konstantin Borisov, Robert - - PowerPoint PPT Presentation

Inexpensive Biosensors based

• n 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

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

• n 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

• f pregnancy tests

and knowledge of terminal illness

Analyte Detection

Transcriptional activation in cell-free extract

Signal Processing

Amplification/Quenching

Signal Detection

Fluorescent proteins/ Colorigenic substrates

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. coli

BL21 prior to lysis

Creation of Sensor Extract

Culture E. coli BL21 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 Input

Estrogen Input

pT7

X

GFP

pT7 Activated T7*

GFP

T7*

X

GFP T7* Activated T7* T7*

Estrogen Sensing System

No Estrogen Input

Estrogen Input

pT7

X

GFP

pT7 Activated T7*

GFP

T7*

X

GFP 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

RNAPX

X

GFP

No Protease Input Protease Input pProt Protease pProt

GFP

GFP

• E. coli

RNAP

Protease Sensing System

pProt

• E. coli

RNAPX

X

GFP

No Protease Input Protease Input pProt Protease pProt

GFP

GFP

• E. 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

DBD

TP TP

p3H

Activation Domain

GFP

GFP DBD

TP

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

X

VEGF-A

VEGF-A Input

VEGF-A detection

Activation Domain Activation Domain

Proteins in Sensor Extract

Estrogen Sensor Preliminary Results

100 200 300 400 500 600 700 800 20 40 60 80 100 120

GFP Fluorescence (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