présentation powerpoint2013.igem.org/files/presentation/paris_saclay.pdf · expression of all the...

2

3

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

4

And you ?

5

PCBs

6

PCBs

•A family of synthetic compounds •High chemical and thermal stability

7

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

8

• Produced and used during the 2nd half of the 20th century

400 million tons produced worldwide

Production of PCBs

9

Incr

easi

ng

PCB

co

nce

ntr

atio

n

10

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

11

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)

13

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

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

17

1

2

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

34

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]

36

HSIM reactions

A + B → C + D [P]

A → B + C [P]

37

Random walk simulation scheme

A

38

Random walk simulation scheme

A

B

39

A

B

A+B → C+D REACTION PROBABILITY P ?

40

Random walk simulation scheme

Random walk simulation scheme

C

D

A+B → C+D

41

42

FNR

Bound

FNR O2

Biological system

Computer model

FNR

Modeling our oxygen sensor

43

FNR

Bound

FNR O2

FNR + O2

Biological system

Computer model

FNR

FNR

Modeling our oxygen sensor

44 44

FNR

Bound

FNR O2

FNR + O2

FNR + Bound

FNR

Biological system

Computer model

FNR

FNR

Modeling our oxygen sensor

45 45 45

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)

47

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 !

A lighter HSIM simulator, wiki-integrated !

52

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

A lighter HSIM simulator, wiki-integrated !

Human Practices

Perception of our project by the public

Safety Open Source

62

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

63

Safety questions

Intellectual Property

64

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! »

66

PS PCR : open source thermo cycler

67

Open Source Reflection

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

71

Electrolab.fr

PS-PCR : thermal block

72

PS-PCR : control electronics

73

PS-PCR : computer software

74

Open source control software, in collaboration with Pierre-Yves

PS-PCR : minimal cost

75

Test

PS-PCR

15µL

76

Conclusion

PbphR1

PbphA1

Thank you !

Super advisors

Claire Nguyên Solenne Sylvie Jean-Luc Philippe