2 3 62% of the people we interviewed are totally ignorant about - PowerPoint PPT Presentation

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62% of the people we interviewed are totally ignorant about this danger 4

And you ? 5

PCBs 6

PCBs P oly C hloro B iphenyls • A family of synthetic compounds • High chemical and thermal stability 7

Utilisation of PCBs • Mostly used as insulating fluids in electrical equipment • But also in coolants, paints and cements, as stabilizing additives in flexible PVC, pesticides… 8

Production of PCBs • Produced and used during the 2 nd half of the 20 th century 400 million tons produced worldwide 9

PCB toxicity Increasing PCB concentration Water and soil contamination 10

PCB toxicity Most PCBs are : ‒ Carcinogenic Increasing PCB concentration ‒ Endocrine disruptors Dangerous for human health and the environment Water and soil contamination Ban of PCBs production and use in France in 1987 11

PCBs in the environment • Contaminated soils and waters (sediments) Paris 28% of French population : Higher exposure than PTMI (Provisional Tolerable Monthly Intake) Sites polluted by PCBs in France 12 Ref : Association Robin des Bois, from data from Ineris, www.pollutionsindustrielles.ecologie.gouv.fr

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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 14

2 step PCB degradation ANAEROBIC PATHWAY (e.g. Dehalobium chlorocoercia ) Highly chlorinated PCBs Lowly chlorinated PCBs Dechlorination AEROBIC PATHWAY (e.g. Burkholderia xenovorans LB 400 ) Lowly chlorinated PCBs Acetyl coenzyme A Oxidative degradation

PCB degradation pathways chosen ANAEROBIC PATHWAY (e.g. Dehalobium chlorocoercia ) tceA AEROBIC PATHWAY (e.g. Burkholderia xenovorans LB 400 ) A1 A2 orf3 A3 A4 B C

Objectives of the PCB Busters ’ project To combine the anaerobic and aerobic degradation 1 pathways in a single bacterium Choice of the chassis: E. coli -Quick growth -Dissemination control -Aerobic and anaerobic metabolisms 2 To construct a PCB sensor system 17

PCB sensor system Objective: build bacteria strain able to detect PCBs in water

PCB sensor system In a Pseudomonas pseudoalcaligenes strain: PCB PCB BphR2 BphR2 Inactive Active PCB BphR2 PbphR1 BBa_K1155001 PbphA1 Bba_K1155002

PCB sensor system PCB Blue signal BphR2 lacZ PbphR1 bphR2 Constitutive PbphA1 promoter +PCB BphR2 is active Blue signal -PCB BphR2 is inactive No signal

PCB sensor system BphR2 lacZ PbphR1 bphR2 Constitutive PbphA1 promoter +PCB BphR2 is active Blue signal -PCB BphR2 is inactive No signal

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: O 2 FNR FNR Active Inactive FNR Pndh* BBa_K1155000 PnirB BBa_K1155004 PnarG BBa_K1155005 PnarK BBa_K1155006

Expression of all the PCB degradation enzymes in E. coli FNR Enzyme fnr PnarK Reductive Constitutive PnarG dechlorination promoter PnirB -O 2 FNR is active Reductive Dechlorination No +O 2 FNR is inactive No Oxidative degradation

Expression of all the PCB degradation enzymes in E. coli FNR Enzyme fnr PnarK Reductive Oxidative Pndh* Constitutive PnarG dechlorination degradation promoter PnirB -O 2 FNR is active Reductive Dechlorination No +O 2 FNR is inactive No Oxidative degradation

Expression of all the PCB degradation enzymes in E. coli FNR O 2 fnr PnarK Reductive Constitutive PnarG dechlorination promoter PnirB -O 2 FNR is active Reductive Dechlorination No +O 2 FNR is inactive No Oxidative degradation

Expression of all the PCB degradation enzymes in E. coli FNR O 2 Enzymes fnr PnarK Reductive Oxidative Pndh* Constitutive PnarG dechlorination degradation promoter PnirB -O 2 FNR is active Reductive Dechlorination No +O 2 FNR is inactive No Oxidative degradation

Testing the system Reporting gene AmilCP (Uppsala 2011 - BBa_K592009) AmilCP

Testing the system Reporting gene AmilCP (Uppsala 2011 - BBa_K592009) AmilCP Term RBS

Testing the system FNR fnr RBS AmilCP Pndh* Term Constitutive promoter -O 2 FNR is active Pndh* repressed No +O 2 FNR is inactive Pndh* no repressed Violet signal

Pndh* - amilCP expression ANAEROBIC CONDITIONS AEROBIC CONDITIONS

Summary Work done: Work in progress: • three FNR activated • Finish the sensor system promoters • Characterize NarG, NarK • one FNR repressed promoter and NirB promoters • Improved a reporter system • Finish the construction of the degradation system • Characterize Pdnh* promoter by using RBS_AmilCP_Term construction

Modeling The O 2 switching system 34

HSIM Patrick Amar and Loïc Paulevé. HSIM: an hybrid stochastic simulation system for 35 systems biology, Electronic Notes in Theoretical Computer Science

HSIM reactions A → B + C [P] 36

HSIM reactions A → B + C [P] A + B → C + D [P] 37

Random walk simulation scheme A 38

Random walk simulation scheme A B 39

Random walk simulation scheme A B A+B → C+D REACTION PROBABILITY P ? 40

Random walk simulation scheme C D A+B → C+D 41

Modeling our oxygen sensor FNR Bound O 2 FNR FNR Biological system Computer model 42

Modeling our oxygen sensor FNR Bound O 2 FNR FNR Biological system Computer model FNR FNR + O 2 43

Modeling our oxygen sensor FNR Bound O 2 FNR FNR Biological system Computer model FNR FNR + O 2 Bound FNR + FNR 44 44

Modeling our oxygen sensor FNR Bound O 2 FNR FNR Biological system Computer model FNR FNR + O 2 Bound FNR + FNR + Bound FNR FNR 45 45 45

Modeling our oxygen sensor FNR Bound O 2 FNR FNR Biological system Computer model FNR FNR + O 2 Bound FNR + FNR + Bound FNR FNR Bound Bound FNR + FNR

Results Green reporter (lack of oxygen) Red reporter (oxygen present) FNR bound to promoter sites 47

A lighter HSIM simulator, wiki-integrated ! 48

A lighter HSIM simulator, wiki-integrated ! 49

A lighter HSIM simulator, wiki-integrated ! 50

A lighter HSIM simulator, wiki-integrated ! 51

A lighter HSIM simulator, wiki-integrated ! 52

A lighter HSIM simulator, wiki-integrated ! 53

A lighter HSIM simulator, wiki-integrated ! 54

A lighter HSIM simulator, wiki-integrated ! 55

A lighter HSIM simulator, wiki-integrated ! 56

A lighter HSIM simulator, wiki-integrated ! 57

A lighter HSIM simulator, wiki-integrated ! 58

A lighter HSIM simulator, wiki-integrated ! 59

A lighter HSIM simulator, wiki-integrated ! 60

Human Practices Perception of our project by the public Safety Open Source 62

Perception of our project by the public Yes What do you think about No our PCBbuster project ? Have you ever heard In favor about PCBs ? Against No opinion 63

Perception of our project by the public Safety questions Intellectual Property 64

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! » 66

Open Source Reflection PS PCR : open source thermo cycler 67

PS-PCR : heating/cooling 68

PS-PCR : heating/cooling  Peltier element (3.78 € ) 69

PS-PCR : heating/cooling  Peltier element (3.78 € )  Heat sink 70

PS-PCR : sample holder Electrolab.fr 71

PS-PCR : thermal block 72

PS-PCR : control electronics 73

PS-PCR : computer software Open source control software, in collaboration with Pierre-Yves 74

PS-PCR : minimal cost 75

Test PS-PCR 15µL 76

Conclusion PbphR1 PbphA1

Thank you ! Super advisors Claire Nguyên Solenne Sylvie Jean-Luc Philippe