présentation powerpoint2013.igem.org/files/presentation/paris_saclay.pdf · expression of all the...
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62% of the people we interviewed are totally ignorant about this danger
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And you ?
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PCBs
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PCBs
•A family of synthetic compounds •High chemical and thermal stability
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PolyChloro Biphenyls
• Mostly used as insulating fluids in electrical equipment
• But also in coolants, paints and cements, as stabilizing
additives in flexible PVC, pesticides…
Utilisation of PCBs
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• Produced and used during the 2nd half of the 20th century
400 million tons produced worldwide
Production of PCBs
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Incr
easi
ng
PCB
co
nce
ntr
atio
n
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Water and soil contamination
PCB toxicity
Incr
easi
ng
PCB
co
nce
ntr
atio
n
Most PCBs are : ‒ Carcinogenic ‒ Endocrine disruptors
Dangerous for human health and the environment
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Water and soil contamination
PCB toxicity
Ban of PCBs production and use in France in 1987
PCBs in the environment
• Contaminated soils and waters (sediments)
Sites polluted by PCBs in France
Paris
12 Ref : Association Robin des Bois, from data from Ineris, www.pollutionsindustrielles.ecologie.gouv.fr
28% of French population : Higher exposure than PTMI
(Provisional Tolerable Monthly Intake)
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Are PCBs biodegradable ?
First PCB degrading bacteria discovered here!
Hudson river
• Bacterial communities can degrade PCBs • Bacteria degrading PCBs have been isolated and studied • But none of them can totally degrade PCBs
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Oxidative degradation
2 step PCB degradation
Lowly chlorinated PCBs Acetyl coenzyme A
(e.g. Burkholderia xenovorans LB 400)
AEROBIC PATHWAY
Dechlorination
Highly chlorinated PCBs Lowly chlorinated PCBs
(e.g. Dehalobium chlorocoercia)
ANAEROBIC PATHWAY
(e.g. Burkholderia xenovorans LB 400)
AEROBIC PATHWAY
(e.g. Dehalobium chlorocoercia)
tceA
C B A4 A3 orf3 A2 A1
PCB degradation pathways chosen
ANAEROBIC PATHWAY
Objectives of the PCB Busters’ project
To combine the anaerobic and aerobic degradation pathways in a single bacterium
To construct a PCB sensor system
Choice of the chassis: E. coli -Quick growth -Dissemination control -Aerobic and anaerobic metabolisms
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PCB sensor system
Objective: build bacteria strain able to detect PCBs in water
PCB sensor system
In a Pseudomonas pseudoalcaligenes strain:
BphR2
PbphR1 BBa_K1155001
PbphA1 Bba_K1155002
PCB
BphR2
Inactive
BphR2
PCB
PCB
Active
PCB sensor system
lacZ bphR2 Constitutive
promoter
PbphR1
PbphA1
+PCB BphR2 is active Blue signal
-PCB BphR2 is inactive No signal
Blue signal BphR2
PCB
PCB sensor system
lacZ bphR2 Constitutive
promoter
PbphR1
PbphA1
+PCB BphR2 is active Blue signal
-PCB BphR2 is inactive No signal
BphR2
PCB degradation system
Objective: build bacteria able to completely degrade PCBs.
PCB degradation system
It involves two pathways:
- Anaerobic: reductive dechlorination
- Aerobic: oxidative degradation
- No bacteria are able to perform both pathways
Objective:
To combine aerobic and anaerobic steps of PCB degradation in E.coli
PCB degradation system
In a E. coli strain:
FNR
PnirB BBa_K1155004
PnarG BBa_K1155005
PnarK BBa_K1155006
FNR
Inactive
O2
Pndh* BBa_K1155000
FNR
Active
Expression of all the PCB degradation enzymes in E. coli
Reductive
dechlorination
fnr Constitutive
promoter
PnarK
PnarG
PnirB
FNR
Enzyme
-O2 FNR is active Reductive Dechlorination No
+O2 FNR is inactive No Oxidative degradation
Expression of all the PCB degradation enzymes in E. coli
-O2 FNR is active Reductive Dechlorination No
+O2 FNR is inactive No Oxidative degradation
Reductive
dechlorination
FNR
fnr Constitutive
promoter
PnarK
PnarG
PnirB
Pndh* Oxidative
degradation
Enzyme
Expression of all the PCB degradation enzymes in E. coli
-O2 FNR is active Reductive Dechlorination No
+O2 FNR is inactive No Oxidative degradation
Reductive
dechlorination
fnr Constitutive
promoter
PnarK
PnarG
PnirB
FNR
O2
Expression of all the PCB degradation enzymes in E. coli
-O2 FNR is active Reductive Dechlorination No
+O2 FNR is inactive No Oxidative degradation
Reductive
dechlorination
fnr Constitutive
promoter
PnarK
PnarG
PnirB
Pndh* Oxidative
degradation
FNR
O2 Enzymes
Testing the system
Reporting gene AmilCP (Uppsala 2011 - BBa_K592009)
AmilCP
Testing the system
Reporting gene AmilCP (Uppsala 2011 - BBa_K592009)
AmilCP RBS Term
AmilCP
FNR
fnr Constitutive
promoter
Pndh*
-O2 FNR is active Pndh* repressed No
+O2 FNR is inactive Pndh* no repressed Violet signal
RBS Term
Testing the system
AEROBIC CONDITIONS ANAEROBIC CONDITIONS
Pndh* - amilCP expression
Summary
Work in progress:
• Finish the sensor system
• Characterize NarG, NarK and NirB promoters
• Finish the construction of the degradation system
Work done:
• three FNR activated promoters
• one FNR repressed promoter
• Improved a reporter system
• Characterize Pdnh* promoter by using RBS_AmilCP_Term construction
Modeling
The O2 switching system
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HSIM
Patrick Amar and Loïc Paulevé. HSIM: an hybrid stochastic simulation system for
systems biology, Electronic Notes in Theoretical Computer Science 35
HSIM reactions
A → B + C [P]
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HSIM reactions
A + B → C + D [P]
A → B + C [P]
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Random walk simulation scheme
A
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Random walk simulation scheme
A
B
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A
B
A+B → C+D REACTION PROBABILITY P ?
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Random walk simulation scheme
Random walk simulation scheme
C
D
A+B → C+D
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FNR
Bound
FNR O2
Biological system
Computer model
FNR
Modeling our oxygen sensor
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FNR
Bound
FNR O2
FNR + O2
Biological system
Computer model
FNR
FNR
Modeling our oxygen sensor
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FNR
Bound
FNR O2
FNR + O2
FNR + Bound
FNR
Biological system
Computer model
FNR
FNR
Modeling our oxygen sensor
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FNR
Bound
FNR O2
FNR + O2
FNR + Bound
FNR FNR + Bound
FNR
Biological system
Computer model
FNR
FNR
Modeling our oxygen sensor
FNR
Bound
FNR O2
FNR + O2
FNR + Bound
FNR FNR + Bound
FNR
Bound
FNR
Bound
FNR +
Biological system
Computer model
FNR
FNR
Modeling our oxygen sensor
Results
Red reporter
(oxygen present)
FNR bound
to promoter sites
Green reporter
(lack of oxygen)
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
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A lighter HSIM simulator, wiki-integrated !
Human Practices
Perception of our project by the public
Safety Open Source
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Perception of our project by the public
What do you think about our PCBbuster project ?
Have you ever heard about PCBs ?
No
Yes
In favor
Against
No opinion
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Safety questions
Intellectual Property
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Perception of our project by the public
PS Bacterial Filter
Gemote system
Sensor detection
Anaerobic and aerobic combination 65
Open Source Reflection
Meetings : - Comparison free software/synthetic biology - Bioterrorism history - Democratization of science - Bioeconomy - Biohacking
Interview : Ellen Jorgensen « I think that people are curious » « DNA is everywhere » « Synthetic biology is very powerful! »
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PS PCR : open source thermo cycler
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Open Source Reflection
PS-PCR : heating/cooling
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PS-PCR : heating/cooling
Peltier element (3.78€)
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PS-PCR : heating/cooling
Peltier element (3.78€)
Heat sink
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PS-PCR : sample holder
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Electrolab.fr
PS-PCR : thermal block
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PS-PCR : control electronics
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PS-PCR : computer software
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Open source control software, in collaboration with Pierre-Yves
PS-PCR : minimal cost
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Test
PS-PCR
15µL
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Conclusion
PbphR1
PbphA1
Thank you !
Super advisors
Claire Nguyên Solenne Sylvie Jean-Luc Philippe