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

Biofilm Killer BIOSCIENCES LYON iGEM TEAM 2012

Context iGEM Synthetic Biology for Innovative Solutions competition

Context Industrial biofilm issues Oil industry : Osli, Schlumberger Cosmetics: Gatefossé Enzyme-based cleaning solutions: Realco

Context � Industry / Manufacturing Unwanted biofilms Final Product Quality Cleaning Costs Use of Chemicals

Context � Existing solutions Mechanical Chemical Enzymes Use of Chemicals � 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

Bacillus subtilis « TORPEDO » Context Project Houry, Gohar, Deschamps, Tischenko, Aymerich, Gruss & Briandet , Proc Natl Acad Sci U S A, 2012.

Context Project KILL constitutive Option Option STICK COAT

Context Project KILL Destroy biofilms

Context Project WEAPON TARGET Staphylococcus strains Biofilm Killer : Bacillix

Context Project KILL Constitutive module Lysostaphin Dispersin

Biocide and scattering agents secreted inside the biofilm and not above, thanks to the swimming capacity of Bacillus .

MECHANISM OF ACTION OF LYSOSTAPHIN Context Project KILL constitutive 2: Lysostaphin Binding and lysis 1: Lysostaphin production Bacillus subtilis S.aureus Lysostaphin

MECHANISM OF ACTION OF DISPERSIN Context Project KILL constitutive 1: Dispersin production Bacillus subtilis 2: Scattering Effect Dispersin S.aureus

Experimental Context Project work KILL constitutive Lysostaphin Dispersin 2 mechanisms of action Complementary action

LYSOSTAPHIN BioBrick™ Experimental Context Project work BBa_K802000 KILL constitutive RBSb Lyso E X S P pBKL25 Er R Lysostaphin gene from Staphylococcus simulans

LYSOSTAPHIN BioBrick™ Experimental Context Project work BBa_K802000 KILL constitutive 24 h Staphylococcus biofilm Upon contact with Bacillus culture for 24 h Observation of Staphylococcus biofilm with Confocal Laser Scanning Microscope

LYSOSTAPHIN BioBrick™ Experimental Context Project work BBa_K802000 KILL CONTROL LYSOSTAPHIN constitutive •Biofilm S. aureus •Biofilm S. aureus •Treated with B. subtillis /empty shuttle plasmid •Treated with B. subtillis /shuttle plasmid + BioBrick™ 30 µm � BIOCIDE EFFECT OF LYSOSTAPHIN

DISPERSIN BioBrick™ Experimental Context Project work BBa_K802001 KILL constitutive Disp RBSb E X S P pBKH26 Er R Dispersin gene from Aggregatibacter actinomycetemcomittans

DISPERSIN BioBrick™ Experimental Context Project work BBa_K802001 KILL constitutive 24 h Staphylococcus biofilm Upon contact with Bacillus culture for 24 h Additional washing step : remove Bacillus culture and add growth medium Observation of Staphylococcus biofilm with Confocal Laser Scanning Microscope BEFORE and AFTER Washing step

DISPERSIN BioBrick™ Experimental Context Project BBa_K802001 work KILL BEFORE washing constitutive DISPERSIN CONTROL •Biofilm S. aureus •Biofilm S. aureus •Treated with B. subtillis /empty shuttle plasmid •Treated with B. subtillis /shuttle plasmid + BioBrick™

DISPERSIN BioBrick™ Experimental Context Project BBa_K802001 work KILL AFTER washing constitutive DISPERSIN CONTROL •Biofilm S. aureus •Biofilm S. aureus •Treated with B. subtillis /empty shuttle plasmid •Treated with B. subtillis /shuttle plasmid + BioBrick™ � SCATTERING EFFECT OF DISPERSIN

Experimental Context Project work KILL constitutive LYSOSTAPHIN + DISPERSIN

Experimental LYSOSTAPHIN + DISPERSIN Context Project work KILL BEFORE washing constitutive LYSOSTAPHIN + DISPERSIN CONTROL

Experimental KILL LYSOSTAPHIN + DISPERSIN Context Project work constitutive AFTER washing CONTROL LYSOSTAPHIN + DISPERSIN LYSOSTAPHIN 30 µm DISPERSIN � BOTH : BETTER RESULT, CLEANER SURFACE

Experimental Context Project work KILL constitutive Option Option COAT STICK Toggle switch

Experimental SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009 Context Project work Avoid new biofilm Option Option COAT STICK formation P xyl RBSb RBSb RBSb sfp abrB lacI

Experimental SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009 Context Project work P xyl Option Option RBSb RBSb RBSb sfp abrB lacI COAT STICK oil Emulsion VORTEX Filtered Filtered S S Bacillus culture Observation 24 h later

Experimental SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009 Context Project work P xyl Option Option RBSb RBSb RBSb sfp abrB lacI COAT STICK Positive control: Supernatant Supernatant LB + SDS BBa_K802009 empty plasmid � Surfactin production

Experimental SURFACTIN generator + BIOFILM repressor + regulator / BBa_K802009 Context Project work P xyl Option Option RBSb RBSb RBSb sfp abrB lacI COAT STICK ∆ ∆ abrB ∆ ∆ ∆ ∆ ∆ ∆ abrB/ BBa_K802009 WILD TYPE Biofilm No biofilm No biofilm � abrB is functional in our part

Experimental Context Project Modelling work

Experimental Context Project Modelling work What do we need for a model? Phenomenon Option Option STICK COAT

Experimental Context Project Modelling work Formal system

Experimental Context Project Modelling work Objectives

Experimental Context Project Modelling work Data and Knowledge

Experimental Context Project Modelling work P xyl sfp sfp abrB abrB lacI lacI XYLOSE xylR xylR P lac

Experimental Context Project Modelling work COAT COAT Surfactant Biofilm (planktonic cells) regulator repressor (planktonic cells) P xyl RNA sfp abrB lacI pol + XYLOSE

Experimental Context Project Modelling work P xyl sfp abrB lacI IPTG xylR P lac

Experimental Context Project Modelling work No surfactant, no biofilm repressor P xyl sfp abrB lacI IPTG + RNA STICK STICK xylR pol (biofilm) (biofilm) P lac

Experimental Context Project Modelling work Modelling objective: Predict the “Biofilm Killer” behavior depending on the inducers’ concentrations

Experimental Context Project Modelling work Physiological Physiological response response Stick Coat Coat Stick IPTG concentration Xylose concentration

Experimental Context Project Modelling work KILL constitutive IPTG induction Xylose induction Option Option STICK COAT

Experimental Context Project Modelling Industrialisation work Clean surfaces Health and food industry Protect surfaces Poultry and animal farming , Chemical industry Oil industry COAT STICK Protective biofilm

Experimental Human Context Project Modelling Industrialisation work Practices INDUSTRIALIST

Experimental Human Context Project Modelling Industrialisation work Practices Which economic system for synthetic biology? E. OSTROM J. STIZGLITZ 2009 Nobel Prize 2001 Nobel Prize

Experimental Human Context Project Modelling Industrialisation work Practices From the Commons to the open source Open source Commons, open source forerunner

Experimental Human Context Project Modelling Industrialisation work Practices What is a commons ? Private sector Public sector Market State Commons Community Members « a jointly owned legal set of rights » Organised by its members

Experimental Human Context Project Modelling Industrialisation work Practices Which economic system for synthetic biology ? A commons as a solution ? Do you think BioBricks TM SHOULD be patentable ? YES NO UNDECIDED

Experimental Human Context Project Modelling Industrialisation work Practices Why not define together the rules of a synthetic biology commons for iGEM ?

Main Achievements � A promising solution to control biofilms on closed surfaces � A proposition for an Economic System for synthetic biology (Collaboration with UBC) � Characterization of new parts : � LYSOSTAPHIN BioBrick™ /BBa_K802000 � DISPERSIN BioBrick™/BBa_K802001 � SURFACTIN + BIOFILM / BBa_K802009 � 2 shuttle plasmids BBa_K802003 and BBa_K802004

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 :