inexpensive biosensors based on cell-free ... -...
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Inexpensive Biosensors based on Cell-Free Extracts
Pitt iGEM 2015Konstantin Borisov, Robert Donahue, Garrett Green, Apurva Patil, Alexander Szul
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InspirationPaper-based tests are currently used sparingly:• pH paper• pregnancy tests• glucose meters
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The media of paper has huge advantages:
Inexpensive cost of production
Simple storage and transportation
Does not require use of laboratory equipment
Inspiration
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The idea:
Could Paper-based Sensors Detect:
Diseases: HIV, Malaria, Salmonella, Cancer
Pollutants: Hormones, Heavy metals, Environmental pharmaceutical pollutants
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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.
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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
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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
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AnalyteDetection
Transcriptional activation in cell-free extract
Signal Processing
Amplification/Quenching
Signal Detection
Fluorescent proteins/ Colorigenicsubstrates
Investigative Methodology
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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
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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
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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
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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
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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*
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Estrogen Sensing System
No Estrogen InputEstrogen Input
pT7
X
GFP
pT7
Activated
T7*
GFP
T7*
XGFP
T7*
Activated
T7*T7*
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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
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Protease Sensing System
pProt
E. coli
RNAP
X XGFP
No Protease InputProtease Input
pProt
Protease
pProt GFP
GFPE. coli
RNAP
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Protease Sensing System
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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
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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.
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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
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Anti-MUC1 antibody detection
Three-Hybrid Versatile System
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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
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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
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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
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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
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Hairpin Quenching Results
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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
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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
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Thank you!Questions?
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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
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Proteins in Sensor Extract
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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