Biofilm Killer BIOSCIENCES LYON iGEM TEAM 2012 Context iGEM - - PowerPoint PPT Presentation

Biofilm Killer

BIOSCIENCES LYON iGEM TEAM 2012

iGEM competition

Context

Synthetic Biology for Innovative Solutions

Industrial biofilm issues

Oil industry : Osli, Schlumberger Cosmetics: Gatefossé Enzyme-based cleaning solutions: Realco

Context

Context

Cleaning Costs Final Product Quality Use of Chemicals

Unwanted biofilms

Industry / Manufacturing

Existing solutions

Context

Chemical Enzymes

Use of Chemicals

Mechanical

Impact on WWTP efficiency Impact on Ecosystems Registry, Evaluation, Authorisation

and restriction of Chemicals (REACh)

Context

Objective: Use synthetic biology to reduce the quantity of chemicals used in cleaning processes

Context Project

Bacillus subtilis « TORPEDO »

Houry, Gohar, Deschamps, Tischenko, Aymerich, Gruss & Briandet, Proc Natl Acad Sci U S A, 2012.

Context Project

KILL

constitutive Option

STICK

Option

COAT

Context Project

KILL

Destroy biofilms

Context Project

WEAPON

Biofilm Killer : Bacillix

TARGET

Staphylococcus strains

Context Project

Lysostaphin Dispersin

KILL

Constitutive module

Biocide and scattering agents secreted

inside the biofilm and not above,

thanks to the swimming capacity of Bacillus.

Context Project

MECHANISM OF ACTION OF LYSOSTAPHIN

1: Lysostaphin production

Bacillus subtilis

Lysostaphin S.aureus

2: Lysostaphin Binding and lysis

KILL

Context Project

MECHANISM OF ACTION OF DISPERSIN

1: Dispersin production

Bacillus subtilis

Dispersin S.aureus

2: Scattering Effect

KILL

Context Project Experimental work

Lysostaphin Dispersin

2 mechanisms of action Complementary action

KILL

Context Project Experimental work

LYSOSTAPHIN BioBrick™ BBa_K802000 Lyso

RBSb

ErR

E X S P

pBKL25 Lysostaphin gene from Staphylococcus simulans KILL

Context Project Experimental work

24 h Staphylococcus biofilm Upon contact with Bacillus culture for 24 h

LYSOSTAPHIN BioBrick™ BBa_K802000 Observation of Staphylococcus biofilm with Confocal Laser Scanning Microscope

KILL

Context Project Experimental work

• Biofilm S. aureus
• Treated with B. subtillis/empty shuttle plasmid

30 µm

CONTROL LYSOSTAPHIN LYSOSTAPHIN BioBrick™ BBa_K802000

• Biofilm S. aureus
• Treated with B. subtillis/shuttle plasmid +

BioBrick™

BIOCIDE EFFECT OF LYSOSTAPHIN

KILL

Context Project Experimental work

DISPERSIN BioBrick™ BBa_K802001

ErR

E X S P

pBKH26 Disp

RBSb

Dispersin gene from Aggregatibacter actinomycetemcomittans KILL

Context Project Experimental work

Additional washing step :

remove Bacillus culture and add growth medium

DISPERSIN BioBrick™ BBa_K802001

24 h Staphylococcus biofilm Upon contact with Bacillus culture for 24 h

Observation of Staphylococcus biofilm with Confocal Laser Scanning Microscope BEFORE and AFTER Washing step

KILL

Context Project Experimental work

BEFORE washing

DISPERSIN BioBrick™ BBa_K802001

• Biofilm S. aureus
• Treated with B. subtillis/empty shuttle plasmid

CONTROL DISPERSIN

• Biofilm S. aureus
• Treated with B. subtillis/shuttle plasmid +

BioBrick™

KILL

Context Project Experimental work

DISPERSIN BioBrick™ BBa_K802001

SCATTERING EFFECT OF DISPERSIN

AFTER washing

KILL

• Biofilm S. aureus
• Treated with B. subtillis/empty shuttle plasmid

CONTROL DISPERSIN

• Biofilm S. aureus
• Treated with B. subtillis/shuttle plasmid +

BioBrick™

Context Project Experimental work

LYSOSTAPHIN + DISPERSIN

KILL

Context Project Experimental work

LYSOSTAPHIN + DISPERSIN CONTROL LYSOSTAPHIN + DISPERSIN BEFORE washing

KILL

Context Project Experimental work

LYSOSTAPHIN + DISPERSIN

BOTH : BETTER RESULT, CLEANER SURFACE

KILL

DISPERSIN LYSOSTAPHIN CONTROL

30 µm

LYSOSTAPHIN + DISPERSIN AFTER washing

KILL

constitutive

Context Project Experimental work

Option

STICK

Option

COAT

Toggle switch

Context Project Experimental work Option

COAT

Option

STICK Avoid new biofilm formation

sfp Pxyl abrB

RBSb RBSb

lacI

RBSb

SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009

Context Project Experimental work

SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009

Filtered S Filtered S

• il

Emulsion VORTEX

Observation 24 h later

Bacillus culture Option

COAT

Option

STICK

sfp Pxyl abrB

RBSb RBSb

lacI

RBSb

Context Project Experimental work

SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009

Positive control: LB + SDS Supernatant BBa_K802009 Supernatant empty plasmid

Surfactin production

Option

COAT

Option

STICK

sfp Pxyl abrB

RBSb RBSb

lacI

RBSb

Context Project Experimental work Option

COAT

Option

STICK

sfp Pxyl abrB

RBSb RBSb

lacI

RBSb

abrB is functional in our part

WILD TYPE No biofilm ∆ ∆ ∆ ∆ abrB Biofilm ∆ ∆ ∆ ∆ abrB/BBa_K802009 No biofilm

SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009

Context Project Experimental work Modelling

Context Project Experimental work Modelling

What do we need for a model?

Phenomenon

Option

STICK

Option

COAT

Context Project Experimental work Modelling

Formal system

Context Project Experimental work Modelling

Objectives

Context Project Experimental work Modelling

Data and Knowledge

Context Project Experimental work Modelling sfp lacI abrB xylR sfp lacI abrB

Pxyl

XYLOSE

xylR

Plac

Context Project Experimental work Modelling sfp lacI abrB Surfactant regulator

Pxyl

RNA pol

COAT

(planktonic cells)

COAT

(planktonic cells) Biofilm repressor

XYLOSE +

Context Project Experimental work Modelling sfp lacI abrB

IPTG

xylR

Pxyl Plac

Context Project Experimental work Modelling sfp lacI abrB

IPTG

xylR

Pxyl Plac

STICK (biofilm) STICK (biofilm) No surfactant, no biofilm repressor

+

RNA pol

Context Project Experimental work Modelling

Modelling objective:

Predict the “Biofilm Killer” behavior depending on the inducers’ concentrations

Context Project Experimental work Modelling

IPTG concentration Physiological response

Stick Coat

Xylose concentration Physiological response

Stick Coat

IPTG induction Xylose induction

KILL

constitutive Option

STICK

Option

COAT

Context Project Modelling Experimental work

Context Project Experimental work Industrialisation

Clean surfaces

Health and food industry Poultry and animal farming , Oil industry Chemical industry

COAT STICK Protective biofilm Modelling

Protect surfaces

Context Project Experimental work Industrialisation Modelling Human Practices

INDUSTRIALIST

Context Project Experimental work Industrialisation Modelling Human Practices

Which economic system for synthetic biology?

• E. OSTROM

2009 Nobel Prize

• J. STIZGLITZ

2001 Nobel Prize

Context Project Experimental work Industrialisation Modelling Human Practices

From the Commons to the open source

Open source Commons, open source forerunner

Context Project Experimental work Industrialisation Modelling Human Practices

What is a commons ?

Private sector Market Public sector State Commons Community Members Organised by its members « a jointly owned legal set of rights »

Context Project Experimental work Industrialisation Modelling Human Practices

A commons as a solution ? Which economic system for synthetic biology ?

Do you think BioBricksTM SHOULD be patentable ? YES NO UNDECIDED

Context Project Experimental work Industrialisation Modelling Human Practices

Why not define together the rules of a synthetic biology commons for iGEM ?

Main Achievements

LYSOSTAPHIN BioBrick™ /BBa_K802000 DISPERSIN BioBrick™/BBa_K802001 SURFACTIN + BIOFILM / BBa_K802009 2 shuttle plasmids BBa_K802003 and BBa_K802004

A proposition for an Economic System for synthetic biology

(Collaboration with UBC)

A promising solution to control biofilms on closed surfaces Characterization of new parts :

Acknowledgments

TEACHING FACILITIES INSA And all scientific, technical and administrative staff from :

• Department of Biosciences of INSA
• MAP and BF2I laboratories
• MICALIS INRA laboratory

And our sponsors :