2 3 62% of the people we interviewed are totally ignorant about [PDF]

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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

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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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Increasing PCB concentration

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Water and soil contamination

PCB toxicity

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Increasing PCB concentration

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

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PCBs in the environment

Contaminated soils and waters (sediments)

Sites polluted by PCBs in France

Paris

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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

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(e.g. Burkholderia xenovorans LB 400)

AEROBIC PATHWAY

(e.g. Dehalobium chlorocoercia)

tceA C B A4 A3

rf3

A2 A1

PCB degradation pathways chosen

ANAEROBIC PATHWAY

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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

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PCB sensor system

In a Pseudomonas pseudoalcaligenes strain:

BphR2 PbphR1 BBa_K1155001 PbphA1 Bba_K1155002

PCB

BphR2

Inactive

BphR2

PCB PCB

Active

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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

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PCB sensor system

lacZ bphR2 Constitutive promoter PbphR1 PbphA1 +PCB BphR2 is active Blue signal

PCB

BphR2 is inactive No signal BphR2

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PCB degradation system

Objective: build bacteria able to completely degrade PCBs.

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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

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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

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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

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Expression of all the PCB degradation enzymes in E. coli

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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

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Expression of all the PCB degradation enzymes in E. coli

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FNR is active Reductive Dechlorination No +O2 FNR is inactive No Oxidative degradation

Reductive dechlorination fnr Constitutive promoter PnarK PnarG PnirB

FNR

O2

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Expression of all the PCB degradation enzymes in E. coli

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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

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Testing the system

Reporting gene AmilCP (Uppsala 2011 - BBa_K592009)

AmilCP

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Testing the system

Reporting gene AmilCP (Uppsala 2011 - BBa_K592009)

AmilCP RBS Term

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AmilCP FNR fnr Constitutive promoter Pndh*

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FNR is active Pndh* repressed No +O2 FNR is inactive Pndh* no repressed Violet signal RBS Term

Testing the system

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AEROBIC CONDITIONS ANAEROBIC CONDITIONS

Pndh* - amilCP expression

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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

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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

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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

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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

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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

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Results

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

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