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