CONTEXT Biofilms are responsible for billions of dollars in production losses and treatment costs in the industry every year:

• food spoilage or poisoning in the food industry ; • pathogens' persistence and dispersal in health industry ; • corrosion in the oil and water industry.

Assuming that the environment is over-saturated with harmful chemicals such as biocides, whose long-term health effects still have to be elucidated, there is a great need for novel solutions to reduce detrimental biofilm effects.

STRATEGY PROJECT Objective: Reduce the use of chemicals in cleaning processes. Solution: Engineer bacterial "torpedos" capable of infiltrating, destroying biofilms (KILL) and protecting the cleaned surfaces by either a surfactant coating (COAT option), or by establishment of a positive biofilm (STICK option).

Advantages: Bacillus strains are non-pathogenic, and do not cause equipment deterioration by corrosion. Supply of active substances within the biofilm should be facilitated by the tunneling activity of Bacillus swimmer cells. This biocide-alternative strategy provides a potential economy, and an environmentally-friendly solution for the control of unwanted biofilms.

HUMAN PRACTICE

After the biofilm destruction: 2 alternatives •Formation of a naturally toxic bio-surfactant and cells remain in planktonic form. •Establishment of a positive biofilm

LacI and XylR: repressors of Plac and Pxyl respectively.

IPTG and Xylose: inducers. In the absence of inducer: both constructions are repressed. IPTG: XylR expressed – No surfactin- No AbrB => Biofilm formation Xylose: XylR inactivation – sfp and abrB transcription => Surfactin production and biofilm repression

LYSOSTAPHIN DISPERSIN Part BBa_K802000 Part BBa_K802001

Constitutive promoter (Pveg) Bacillus RBS lysostaphin gene from S. simulans

THE “LYSOSTAPHIN” PART ENABLES B. subtilis TO EFFICIENTLY KILL S. aureus CELLS THUS CAUSING A

DRASTIC REDUCTION OF THE BIOFILM.

METHOD: S. aureus fluorescent strain RN4220 pALC2084 was used to form biofilms in 96-well microscopic-grade microtiter plates. B. subtilis 168 containing or not the lysostaphin gene in shuttle vector were grown on LB medium and were loaded on S. aureus biofilm. Biofilms were observed under a time-lapse confocal microscope, after incubation at 30°C without shaking.

CONTROL

S. aureus biofilm treated for 24h with B. subtilis / BBa_K802000

METHOD: same as described for lysostaphin with an additional washing step with 250 µL of sterile culture medium.

KILL

LYSOSTAPHIN: bacterial biocide isolated from Staphylococcus simulans and which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan leading to cell lysis.

Cells expressing GFP were excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm using an oil-immersion objective with a magnification of 63x. The overall three-dimensional structures of the biofilms were scanned from the solid surface to the interface with the growth medium, using a step of 1 µm.

DISPERSIN: bacterial scattering agent isolated from A. actinomycetemcomittans and which specifically catalyses hydrolysis of extracellular matrix produced by bacteria.

COMBINED EFFECT

COAT STICK

SURFACTIN + BIOFILM

sfp

Pxyl

abrB RB

Sb

RB

Sb

lacI

RB

Sb

A commons as a solution ?

Which economic system for synthetic biology ?

Private sector Market

Public sector State

Commons Community Members

Organised by its members « a jointly owned legal set of rights »

INSA de LYON – Biosciences iGEM Team 2012

MODELLING

Experimental model:

• Staphylococcus aureus as the detrimental biofilm.

• Bacillus subtilis as the "Biofilm Killer" agent.

Part BBa_K802009

BBa_K802000

S. aureus biofilm treated for 24h with B. subtilis / empty shuttle plasmid

Constitutive promoter (Pveg) Bacillus RBS dispersin gene from A. actinomycetemcomittans Terminator

lysostaphin

RB

Sb

RB

Sb

dispersin

CONTROL BBa_K802001

30 µm

S. aureus biofilm treated for 4h with B. subtilis / empty shuttle plasmid

S. aureus biofilm treated for 4h with B. subtilis / BBa_K802001

THE “DISPERSIN” PART ENABLES B. subtilis TO EFFICIENTLY SCATTER S. aureus CELLS AFTER A WASHING STEP.

BBa_K802000 & BBa_K802001

BOTH: BIOFILM ERADICATION

CLEANER SURFACE

S. aureus biofilm treated for 24h with B. subtilis / BBa_K802000 and B. subtilis / BBa_K802001

WILD-TYPE No biofilm

D abrB Biofilm

D abrB / BBa_K802009 No biofilm

Positive control: Sterile culture medium + SDS EMULSION FORMATION

BBa_K802009: Supernatant from Bacillus / BBa_K802009 + oil EMULSION FORMATION

Negative control: Supernatant from

Bacillus / empty shuttle plasmid + oil

NO EMULSION

SURFACTIN: sfp codes for an enzyme which catalyzes synthesis of lipoprotein surfactin which has amphiphilic properties that affect surface tension of liquids (detergent property).

BIOFILM: abrB codes for a repressor which controls expression of genes involved in biofilm formation.

sfp lacI abrB

Pxyl

Surfactant regulator

Biofilm repressor

Positive Biofilm

Surfactant

IPTG

XYLOSE

xylR Plac

GENE-REGULATORY NETWORK

Construction of the biological model: 2 promoters (Pxyl and Plac), 2 repressors (LacI and XylR proteins) and 2 inducers (IPTG (isopropyl β-D-1-thiogalactopyranoside) and Xylose), and two effector genes sfp and abrB.

TOGGLE SWITCH

•Phenomenon

•Formal system

•Objectives

•Data and Knowledge Difficulties to obtain Bacillus specific data

DNA constructions to obtain new values of parameters needed for differential equations

BBa_K802009

Pxyl

GFP

RB

Sb

Before washing step

After washing step

THE PART ENABLES B. subtilis TO PRODUCE SURFACTIN AND TO CONTROL BIOFILM FORMATION.

Do you think BioBricksTM SHOULD be patentable ?

YES NO UNDECIDED

INDUSTRIALIZATION

Chemical industries COAT STICK

protective biofilm

KILL

1 - Introduction of BIOFILM KILLER 2- 4h cycle in hydraulic system A : tank cleaning 3- 4h cycle in hydraulic system B/C: circuit cleaning 4- water rinsing

5- Introduction of 0.45 and 0.2 µM filters in *

to recover Biological wastes 6- Optional : activate the COAT or STICK options during the cycles

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

Protect surfaces

Health, cosmetic and food industries

Clean surfaces BIOFILMS ISSUES:

+

Poultry and animal farming , Oil industries

Option stick

Option coat