iGEM Final al Jam ambore ree 11/05/201 05/2011 The he Dev - - PowerPoint PPT Presentation

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iGEM Final al Jam ambore ree 11/05/201 05/2011 The he Dev evic ice Mining, People and the Environment, 03/12/2009 2011 World Championship Jamboree 2 The he Dev evic ice Importance to quantify mercury contained in water 2011 World

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iGEM Final al Jam ambore ree 11/05/201 05/2011

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The he Dev evic ice

Mining, People and the Environment, 03/12/2009

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The he Dev evic ice

Importance to quantify mercury contained in water

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The he Dev evic ice

  • Meeting with a Chemical Industry

Industrial Needs:

  • Precise quantification (ECD: 50µg/L)
  • Reliable technics

Partner rtnership ship wit ith

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The he Dev evic ice

State of the art atomic absorption spectrometry

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The he Dev evic ice

Easy to handle Cheap

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  • Specifications :

The he Dev evic ice

Single bacterial strain Comparative measurement Visual result

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  • Single Bacterial Strain

The he Dev evic ice

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The he Dev evic ice

  • Comparative Measurement

– The reference molecule IPTG

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The he Dev evic ice

  • Comparative Measurement

– Emergence of 2 bacterial behaviors :

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The he Dev evic ice

  • Comparative Measurement

– Emergence of 2 bacterial behaviors :

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The he Dev evic ice

  • Visual Result

– At the boundary both senders and receivers interact:

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2-way Switch ch Colorat

  • ration

ion Power wer Button

The he Gen enet etic ic Net etwork rk

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  • 3-level Genetic Network

The he Gen enet etic ic Net etwork work

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  • 2-way Switch

Switch between 2 pathways

The he Gen enet etic ic Net etwork work

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  • 2-way Switch coupled with

Quorum Sensing

The he Gen enet etic ic Net etwork work

1 Pathway 1 Behavior

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The he Gen enet etic ic Net etwork work

  • 2-way Switch coupled with

Quorum Sensing

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The he Gen enet etic ic Net etwork work

  • 2-way Switch coupled with

Quorum Sensing

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2-way Switch ch Colorat

  • ration

ion Power wer Button

Mod

  • del

eling ing an and d Exp xper erim iment ents

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Modeling eling AND Exp xper eriments iments

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  • 2-way Switch

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • 2-way Switch ODEs

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • Evolution of TetR concentration (1mM IPTG)

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • Biological Results

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • Evolution of GFP fluorescence (1mM IPTG)

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • Biological bistability

Mod

  • del

eling ing AND Exp xper erim imen ents

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aTc IPTG

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  • Biological bistability

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • Biological bistability

Mod

  • del

eling ing AND Exp xper erim imen ents

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  • Stochastic study of bistability

Mod

  • del

eling ing AND Exp xper erim imen ents

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Modeling eling AND Exp xper eriments iments

  • Quorum Sensing

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Modeling eling AND Exp xper eriments iments

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Modeling eling AND Exp xper eriments iments

  • Result on the plate

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aTc=50 ng/mL aTc=150 ng/mL

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  • Result on the plate

Modeling eling AND Exp xper eriments iments

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  • Power Button

Modeling eling AND Exp xper eriments iments

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  • Post-transcriptional network

Modeling eling AND Exp xper eriments iments

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  • Post-transcriptional network

Modeling eling AND Exp xper eriments iments

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  • GFP fluorescence obtained with fha

Modeling eling AND Exp xper eriments iments

fha rbs

Fluorescence GFP Counts

background

GFP Fluorescence Counts

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Modeling eling AND Exp xper eriments iments

  • The power switch using RsmA & rsmY

70 103 104 102 101 100

GFP fluorescence

140 210 280 350

Counts

Autofluorescence Fha Fha + RsmA Fha + RsmA + rsmY

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  • Channels on the device

Modeling eling AND Exp xper eriments iments

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  • Channels on the device

Modeling eling AND Exp xper eriments iments

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  • Device specifications

Response time

1 hour

Sensitivity

10-7 M (20µg/L)

10% precision

104 bacteria per channel

Modeling eling AND Exp xper eriments iments

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Interdi erdisc sciplinari narity ty Promot

  • motion

ion

Hum uman an Pra ract ctic ice

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Huma man Pra ractice tice

  • Synthetic Biology

An interdisciplinary approach of living systems !

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  • How to improve the interaction between

Biologists & Modelers ?

Huma man Pra ractice tice

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Huma man Pra ractice tice

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Huma man Pra ractice tice

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  • Test organization

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Huma man Pra ractice tice

(With Flyers) (Without Flyers)

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  • Test results

Huma man Pra ractice tice

2 4 6 8 10

Group A (With Flyers) Group B (Without Flyers)

Average Score (/10)

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Huma man Pra ractice tice

16 16% Negative 84 84% Positive

Did the flyers help you you to better er understand rstand the questi tion

  • ns?

?

  • Test results

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Huma man Pra ractice tice

38 38% Negative 62 62% Positive

Did the flyers help you you to better er interact act with your ur partne ner? ?

  • Test results

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Huma man Pra ractice tice

  • Promoting Synthetic Biology

– Conferences – Media Attention (GreNews/A savoir/Radio)

iGEM Grenoble 2011 at MINATEC iGEM Grenoble 2011 at INRIA

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  • Major achievements

Innovative mercury biosensor Industrial interest ! Proof of concept of 3 modules: 19 biobricks added. Models giving all necessary specifications of the device. New iGEM flyers for biologists & modelers. iGEM Grenoble 2012 is launched!

Conc nclusion lusion

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  • Instructors

Acknowledg knowledgments ments

  • Hans Geiselmann
  • Franz Bruckert
  • Marianne Weidenhaupt
  • Hidde De Jong
  • Delphine Ropers
  • Robert Baptist
  • Advisor
  • Stéphane Pinhal

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Acknowledg knowledgments ments

  • Sponsors

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Acknowledg knowledgments ments

  • Sponsors

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  • « CEA- Direction des Sciences du vivant »
  • « CEA- Direction des Sciences de la Matière »
  • « CEA-Direction de la Recherche Technologique »
  • Programme Transversal Nanotoxicologie
  • Programme Transversal Technologies pour la Santé
  • Programme transversal Nanosciences
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Thank you for your attention Questions ?

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Tr Tran ansla slational tional re regu gulation lation

given IPTG gradient nt with an aTc concentration of 10−6 M Low TetR concen entr tratio ation n : S Switch h at at lower er concent centra ration ion of IPTG (red curve) e). .

Infl fluence uence of f a a tran anslat ation ional al regulat atio ion n system m on the he switch

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Tr Tran ansla slational tional re regu gulation lation

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Infl fluence uence of f RsmA product ction ion on the growth

  • f

f E c coli DH5α?

Tr Tran ansla slational tional re regu gulation lation

  • pVLT31

T31-rsmA rsmA or empty ty

  • No growth

th difference rence

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Fha1 ha1 gene leader er sequence uence mag operon

  • n leader

er sequence uence

Tr Tran ansla slational tional re regu gulation lation

Construc

ructs s used to charact aracter erise se RsmA system em rsmY sRNA sequence uence

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Tr Tran ansla slational tional re regu gulation lation

Flow w cyt ytomet meter er : dot plots of f the 40 40000 bacter eria a analysed ysed

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Tr Tran ansla slational tional re regu gulation lation

Flow w cytomet eter er : first test of the leader er sequences uences

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Tr Tran ansla slational tional re regu gulation lation

Flow w cytomet eter er : compilation tion of controls rols and the two leader er sequence ence signal

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18,2% 0,6% 0% 5% 10% 15% 20% 25% 30% fha-gfp mag-gfp

Tr Tran ansla slational tional re regu gulation lation

Relative ve RBS strength ngth to the reference erence RBS. SD of four indepen pende dent t measurem urement ents. s.

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100% 15% 24%

0% 20% 40% 60% 80% 100% 120%

fha/fha rsmA/fha rsmaA+rsmY/fha × 1,5 ÷ 6,5

Tr Tran ansla slational tional re regu gulation lation

Single, le, double, e, and triple transfor formations.

  • tions. Using RsmA,

, fha-GFP FP signal is much h decreased.

  • reased. Upon transcr

cription tion of rsmY, , fha-GFP FP signal rises. s. Effect ct of RsmA and RsmA + + rsmY on fha-GFP FP signal

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100% 6% 6% 0% 20% 40% 60% 80% 100% 120% fha/fha rsmA/fha rsmaA+rsmY/fha

÷ 17,5 70 103 104 102 101 100

GFP

140 210 280 350

Count

Auto-fluorescence Fha

  • Fha

+ rsmA

  • Fha

+ rsmA + rsmY

  • Tr

Tran ansla slational tional re regu gulation lation

Using values s from the whole e population, ion, RsmA appears rs more important nt, rsmY effect t is shunt nted ed by 30%. Clon

  • ne

e variab ability ty? Effect ct of RsmA and RsmA + + rsmY on fha-GFP FP signal

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1 1 1 1 2 1 3 1 4 G F P

mag mag + rsmA mag + rsmA + rsmY

Tr Tran ansla slational tional re regu gulation lation

Effect ct of RsmA and RsmA + + rsmY on mag-GF GFP P signal Single, le, double, e, and triple transfor formations.

  • tions. mag-GF

GFP P sigla is very low, and totally shut off ff when n bacteria teria contain n rsmA mA.

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Brenic et Lory, 2009

Tr Tran ansla slational tional re regu gulation lation

Cellular ar context ext of f the RsmA mA sys ystem em

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Tr Tran ansla slational tional re regu gulation lation

Bacterial Small RNA Regulators. Nadim Majdalani et al,2005

An other er translat ation ional al regulation ation system em : dsrA rA / / rpoS

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