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A microbial beacon for cancer detection
Primary cancer Metastasis
1 WHO Cancer Fact Sheet No297
A microbial beacon for cancer detection Primary Metastasis - - PowerPoint PPT Presentation
A microbial beacon for cancer detection Primary Metastasis - - PowerPoint PPT Presentation
A microbial beacon for cancer detection Primary Metastasis cancer 1 WHO Cancer Fact Sheet N o 297 Primary Metastasis cancer Late treatment Bad prognosis 1 WHO Cancer Fact Sheet N o 297 Primary Metastasis cancer Late treatment
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Primary cancer
Late treatment Bad prognosis
Metastasis
1 WHO Cancer Fact Sheet No297
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Primary cancer
Late treatment Bad prognosis
Metastasis
General and early
1 WHO Cancer Fact Sheet No297
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Primary cancer
Late treatment Bad prognosis
Metastasis
General and early
Circulating tumor cells in blood
1 WHO Cancer Fact Sheet No297
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Primary cancer
Late treatment Bad prognosis
Metastasis
General and early
Circulating tumor cells in blood
Early treatment Better prognosis
1 WHO Cancer Fact Sheet No297
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Primary cancer
Late treatment Bad prognosis
Metastasis
General and early
Circulating tumor cells in blood
Early treatment Better prognosis
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Early detection
- f circulating tumor
cells (CTCs)
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Early and Universal Diagnosis not yet Reliable
- Inconsistent results by existing
tests
- Not used in clinical practice
Need to find more stable markers
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Early and universal diagnosis not yet reliable
- Inconsistent results by existing
tests
- Not used in clinical practice
Need to find more stable markers
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MicroBeacon detects two general cancer markers
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Marker 1: Elevated Lactate Production Rate by Cancer
3 Cancer cell Non-cancer cell L-Lactate MicroBeacon
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Marker 1: Elevated Lactate Production Rate by Cancer
3 Cancer cell Non-cancer cell L-Lactate MicroBeacon
Signal 1
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Marker 1: Elevated Lactate Production Rate by Cancer
Challenge: Only three-fold[1] difference in production rate!
3 Cancer cell Non-cancer cell L-Lactate MicroBeacon
Signal 1
[1] Anal. Chem. 82.12 (2010): 5082
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Marker 2: Cancer Cell’s sTRAIL Susceptibility
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Death Receptor sTRAIL MicroBeacon Cancer cell Non-cancer cell Phosphatidylserine
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Marker 2: Cancer Cell’s sTRAIL Susceptibility
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Death Receptor sTRAIL MicroBeacon Cancer cell Non-cancer cell Phosphatidylserine
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Marker 2: Cancer Cell’s sTRAIL Susceptibility
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Death Receptor sTRAIL MicroBeacon Cancer cell Non-cancer cell Phosphatidylserine Apoptotic cell
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Marker 2: Cancer Cell’s sTRAIL Susceptibility
Signal 2 Cancer cell Non-cancer cell Phosphatidylserine Death Receptor sTRAIL MicroBeacon
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Two-step Sequential Filtering
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Phosphatidyl- serine Apoptosis Lactate Lactate sensor Density sensor sTRAIL GFP
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Single-cell Analysis Setup for Detecting Scarce CTCs
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Microfluidic chip Blood MicroBeacon
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Microfluidic chip Blood MicroBeacon
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Single-cell Analysis Setup for Detecting Scarce CTCs
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Microfluidic chip Blood MicroBeacon
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Single-cell Analysis Setup for Detecting Scarce CTCs
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Microfluidic chip Blood MicroBeacon
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Single-cell Analysis Setup for Detecting Scarce CTCs
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Single-cell Analysis Setup for Detecting Scarce CTCs
Microfluidic chip Blood MicroBeacon Cancer
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Lactate Production Rate
Marker 1
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Single Cell Model
- System of 10 ODEs and 40
parameters
- Mechanistic assumptions based on
reaction rates
- Characterization of lactate sensor
- Preliminary characterization of
density sensor
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Model Predicts: Simple System Will Not Work
Threshold
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Incoherent Feed-forward Loop (IFFL)[1]
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Threshold
[1] Molec. cell 36.5 (2009): 894-899.
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Implementation of the Lactate Sensor
X Y Z
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Single input promoter controlled by LldR, responsive to lactate Hybrid promoter controlled by LldR and LacI
Two Promoters Needed for Incoherent Feed-forward Loop
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Single input promoter controlled by LldR, responsive to lactate Hybrid promoter controlled by LldR and LacI
Two Promoters Needed for Incoherent Feed-forward Loop
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Single Input Promoter
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Plld
O1 O2 gfp
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Single Input Promoter
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Plld
O1 O2 gfp
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Single Input Promoter: Repression and Activation
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Plld
O1 O2 LldR gfp lactate
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Activation Repression
Single Input Promoter: Repression and Activation
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Plld
O1 O2 LldR gfp lactate
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Design of Promoter Library
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Design of Promoter Library
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Different:
- Core promoter
J23117 O2 O1
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Design of Promoter Library
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Different:
- Core promoter
- Architecture
spacer O1 J23117 O2 J23117 O2 O1
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Design of Promoter Library
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Different:
- Core promoter
- Architecture
- Spacing
J23117 O2 O1 spacer O1 J23117 O2
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BBa_K1847005 BBa_K1847006 BBa_K1847007 BBa_K1847008 BBa_K1847009 BBa_K1847002 BBa_K1847003 BBa_K1847004
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Strength of core promotor Different architecture
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BBa_K1847005 BBa_K1847006 BBa_K1847007 BBa_K1847008 BBa_K1847009 BBa_K1847002 BBa_K1847003 BBa_K1847004
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Strength of core promotor Different architecture
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Increased ON/OFF Ratio
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Plld
O1 O2 LldR gfp lactate
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Model Predicts: Lactate Import by LldP is Important
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Intracellular
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Experiments Confirm: LldP is Essential for Sensing
Plld
O1 O2 LldR gfp Pconst lldP LldP lactate
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Detection System Works in Mammalian - E. coli Co-culture
Jurkat, E. coli PlldR::gfp, lldR Jurkat, E. coli PlldR::gfp, lldR, lldP
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Single input promoter controlled by LldR, responsive to lactate Hybrid promoter controlled by LldR and LacI
Two Promoters Needed for Incoherent Feed-forward Loop
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Hybrid Promoter
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- Fluo. 104 (Au. norm.)
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sTRAIL Sensitivity
Marker 2
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How Can One Detect PS Exposure on the Target Cell?
22 Annexin V- Alexa 488 [1] JBC 272.42: 26159 Phosphatidylserine Apoptotic cancer cell Death receptor Cancer cell
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Annexin V Displayed on Bacterial Outer Membrane
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AD: AutoDisplay[1] An-V: Annexin V
[1] J. Bact. 179,3 794
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Density
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How to Detect E. coli Bound to a Cancer Cell
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Density Quorum sensing
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How to Detect E. coli Bound to a Cancer Cell
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Genetic Design: Quorum Sensing
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Genetic Design: Combined Circuit
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Why is LuxR the Output of the Lactate Sensor?
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Two Additional Models
Compartment model
- Multi-compartment ODE system with
instant diffusion within a compartment
- System design and worst-case testing
3D spatio-temporal model
- Whole system with biological properties
- System of 27 ODEs and PDEs and 42
parameters in multiple domains
- Validation in more realistic environment
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An AND Gate is Insufficient
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AND gate Sequential filtering
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Expected Behavior of MicroBeacon
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Compartment Model: Incoherent Feed-forward Loop is Necessary
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Measurement Measurement
Without Feed-forward With Feed-forward
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3D Spatio-temporal Model
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3D Spatio-temporal Model: MicroBeacon is Reliable
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Achievements
Designed, characterized, and submitted over 17 new biobricks Demonstrated that MicroBeacon detects lactate produced by cancer cells Developed and used 3 different models to understand, evaluate, and optimize our design Collaborated with 3 other iGEM teams Outreach: Interviews, teaching in school, newspapers
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Attributions
Lab, logo design Lab, human practices, poster Lab, poster, safety Lab, organization, sponsoring Modeling, poster,
- rganization, chip
Modeling, logo design, wiki development 35
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Thank you
- Instructors: Sven Panke, Jörg Stelling, Kobi Benenson, Prof.
Savas Tay
- Advisors: Daniel Gerngross, Sabine Österle, Lukas Widmer,
Margaux Dastor, Christian Jordi, Janina Linnik
- Other Support: Erica Montani, Verena Jäggin, Michal Stanczak,
Markus Jeschek, Matthias Mehlig, Myriam Moisan, Tino Frank, Johannes Thoma, Gaspar Morgado, Michael Junkin, Bartolomeo Angelici, Max Endele, Martin Etzrodt
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Sponsors
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