Team:Lyon-INSA-ENS/Project/Context

From 2011.igem.org

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              A project anchored at the heart of current concerns<br><HR>
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            Engineering E. coli adhesion for improved bioremediation<br><HR>
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The activity of modern nuclear power plants with pressurized water reactors generates
+
<b>Biofilms and depollution.</b> Often associated to disease and unwanted surface fouling, biofilms
-
radioactive effluents that contain among other things <b> radioactive cobalt </b>. The tubing of
+
are helpful in bioremediation, biocatalysis or as microbial fuel cells. Bioremediation processes
-
the cooling circuit is made of <b> a steel alloy rich in stable cobalt (<sup>59</sup>Co)</b>. Undergoing neutron
+
use natural microbial ability to degrade organic substances or to modify metal speciation
-
bombardment coming from the reactor, <b>this stable cobalt changes into its radioactive isotope</b>,
+
by immobilization or volatilization. Such properties are observed in natural ecosytems as in
-
cobalt 60 (<sup>60</sup>Co).
+
artificial systems used to clean solid or liquid waste. Intensity and quality of the microbial
 +
activities depend on local physical and chemical factors, and also on the way of life of
 +
microbes (biofilm or plankton). Biofilm formation is associated to resistance to most of
 +
biocides by diverse mechanisms. Adhesion is therefore a choice property in most remediation
 +
processes.
     <br/><br/>
     <br/><br/>
-
The capture of this metal is interesting on a <b>sanitary</b> point of view, because it represents
+
<b>Strategy: boost natural abilities!</b> Binding to extracellular matrix, efflux pumps and
-
<b>a danger under both its radioactive and stable forms (carcinogenic)</b>. It also represents an
+
activation of transporters allow concentration and sequestration of biocides such as metals.
-
advantage on an <b>environmental</b> point of view, in order to avoid contamination of waters, soil
+
Genetic engineering allows to boost these activities and to improve the treatment of metallic
-
and groundwater. Even with a short half life, cobalt 60 emits <b>high intensity gamma rays</b>, and
+
pollution, especially for toxic metals in low concentration. Classic chemical processes using
-
decays to nickel, which is stable but polluting.
+
ion-exchange resins are then economically inappropriate, and thanks to their high selectivity,
 +
micro-organisms appear very efficient.
     <br/><br/>
     <br/><br/>
-
Controlled immobilization of radioactive cobalt is both an important sanitary and environmental
+
<b>OGM biofilters for nuclear liquid waste treatment.</b> Treatment of nuclear waste is a
-
issue, which we intend to solve with <b>an innovative and economical response</b>. A researcher
+
promising application for biological treatment of metals contaminations. Confinement is
-
from the Lyon INSA-ENS team, Agnès Rodrigue, has recently constructed a E.coli strain able
+
indeed a major hindrance to the use of Genetically Modified Organisms for waste treatment.
-
to <b>eliminate 85% of radioactive cobalt (<sup>60</sup>Co)</b>, initially present as traces in a simulated nuclear
+
Since radioactive waste are submitted to a strict and regulated handling, use of GMO in this
-
effluent made up of a mix of heavy metals, in only twice one-hour incubation (Appl Microbio
+
context should be well-accepted by the society. The activity of modern nuclear power plants
-
Biotechnol 2009 81:571- 578).
+
with pressurized water reactors generates radioactive effluents that contain among others
 +
radioactive cobalt. The tubing of the cooling circuit is made of a steel alloy rich in cobalt and
 +
nickel. Under neutrons bombardment coming from the reactor, these stable metals change into
 +
radioactive isotopes. Corrosion results in solubilization of these activation products, and water
 +
contamination.
     <br/><br/>
     <br/><br/>
-
The process that was developed by Agnès Rodrigue’s team ensures the decontamination
+
<b>Selective cobalt capture.</b> Controlled immobilization of radioactive cobalt is both an
-
of cobalt <b>up to 0,5 ppm</b> (8 nM in 100 000L) with <b>only 4kg of bacteria</b> as against 50kg with
+
important sanitary and environmental issue. Activation products are routinely captured
-
an unmodified bacterium or 8,000kg of an ion-exchange polymer. This kind of process with
+
by using synthetic ion exchangers. This generates large volume of solid waste due to the
-
modified bacteria will be a good value because the production of bacteria in a bioreactor is
+
nonspecific nature of ion sorption. In this context, a researcher from the Lyon INSA-ENS
-
rather economical. However, one issue remained unsolved at the end of this study, that is the
+
team has recently constructed an <b>E.coli strain able to eliminate 85% of radioactive cobalt <b/>
-
<b>separation of cobalt-fixing bacteria</b>.
+
initially present as traces in a simulated nuclear effluent. However, the recovery of cobalt-
 +
fixing bacteria has to be facilitated before to consider industrial application.
     <br/><br/>
     <br/><br/>
-
The first objective of our project is, with the most recent genetic engineering techniques, to
+
<b>“Cobalt Buster” biofilter.</b> Our objective is to facilitate the recovery of the metal-stuffed
-
<b>induce the fixation </b> of optimized bacteria for the capture and retention of cobalt in response to the
+
bacteria by inducing their fixation to a solid support. (France 3 movie?). We choose to
-
presence of contaminants in the effluent to be treated.
+
engineer this sought-after adherence property by using the exceptional properties of the
 +
curli amyloid fibers. In a first approach, a synthetic operon comprising the absolutely
 +
required genes for curli production under control of a strong and cobalt-inducible promoter
 +
was designed and synthesized. This construct allows K12 E. coli (MC4100, MG1655,
 +
NM522…) to stick to polystyrene and glass. Adherence is reinforced by the presence of
 +
cobalt and should avoid free floating growth. In a second approach, a part allowing the
 +
constitutive overproduction of the curli superactivator OmpR234 was constructed. By
 +
activating the cryptic curli genes located in the core genome of K12 E. coli, this part allows
 +
to increase bacterial adherence to polystyrene and glass. Such results lead us to discuss of a
 +
possible industrialization with the ASSYSTEM company and of research and development
 +
perspectives with the EDF company.
-
    <br/><br/>
 
-
 
-
A second objective aims to develop a system to construct custom-built “biofilm inducible”
 
-
strains. Our goal is to construct <b>captors able to launch the formation of biofilm in response to
 
-
the presence of various radioactive or regular pollutants</b>, and to offer <b>more efficient</b> and <b>cheaper</b>
 
-
bioremediation processes.
 
-
 
-
    <br/><br/>
 
-
To conclude, our objective is to deposit <b>a part able to make any strains inducible to cobalt</b>. In presence of this element, strains will become adherent and will form biofilm thanks to their curli.
 
     <br/><br/><br/>  
     <br/><br/><br/>  

Revision as of 19:40, 19 September 2011



Stage 1 Stage 1 Stage 2 Stage 3



Overproduction of curli via a synthetic operon controled by a cobalt-inducible promoter Prcn-csgBAEFG













Overexpression of curli genes via the superactivator OmpR234












Engineering E. coli adhesion for improved bioremediation



Biofilms and depollution. Often associated to disease and unwanted surface fouling, biofilms are helpful in bioremediation, biocatalysis or as microbial fuel cells. Bioremediation processes use natural microbial ability to degrade organic substances or to modify metal speciation by immobilization or volatilization. Such properties are observed in natural ecosytems as in artificial systems used to clean solid or liquid waste. Intensity and quality of the microbial activities depend on local physical and chemical factors, and also on the way of life of microbes (biofilm or plankton). Biofilm formation is associated to resistance to most of biocides by diverse mechanisms. Adhesion is therefore a choice property in most remediation processes.

Strategy: boost natural abilities! Binding to extracellular matrix, efflux pumps and activation of transporters allow concentration and sequestration of biocides such as metals. Genetic engineering allows to boost these activities and to improve the treatment of metallic pollution, especially for toxic metals in low concentration. Classic chemical processes using ion-exchange resins are then economically inappropriate, and thanks to their high selectivity, micro-organisms appear very efficient.

OGM biofilters for nuclear liquid waste treatment. Treatment of nuclear waste is a promising application for biological treatment of metals contaminations. Confinement is indeed a major hindrance to the use of Genetically Modified Organisms for waste treatment. Since radioactive waste are submitted to a strict and regulated handling, use of GMO in this context should be well-accepted by the society. The activity of modern nuclear power plants with pressurized water reactors generates radioactive effluents that contain among others radioactive cobalt. The tubing of the cooling circuit is made of a steel alloy rich in cobalt and nickel. Under neutrons bombardment coming from the reactor, these stable metals change into radioactive isotopes. Corrosion results in solubilization of these activation products, and water contamination.

Selective cobalt capture. Controlled immobilization of radioactive cobalt is both an important sanitary and environmental issue. Activation products are routinely captured by using synthetic ion exchangers. This generates large volume of solid waste due to the nonspecific nature of ion sorption. In this context, a researcher from the Lyon INSA-ENS team has recently constructed an E.coli strain able to eliminate 85% of radioactive cobalt initially present as traces in a simulated nuclear effluent. However, the recovery of cobalt- fixing bacteria has to be facilitated before to consider industrial application.

“Cobalt Buster” biofilter. Our objective is to facilitate the recovery of the metal-stuffed bacteria by inducing their fixation to a solid support. (France 3 movie?). We choose to engineer this sought-after adherence property by using the exceptional properties of the curli amyloid fibers. In a first approach, a synthetic operon comprising the absolutely required genes for curli production under control of a strong and cobalt-inducible promoter was designed and synthesized. This construct allows K12 E. coli (MC4100, MG1655, NM522…) to stick to polystyrene and glass. Adherence is reinforced by the presence of cobalt and should avoid free floating growth. In a second approach, a part allowing the constitutive overproduction of the curli superactivator OmpR234 was constructed. By activating the cryptic curli genes located in the core genome of K12 E. coli, this part allows to increase bacterial adherence to polystyrene and glass. Such results lead us to discuss of a possible industrialization with the ASSYSTEM company and of research and development perspectives with the EDF company.





ENS assystem Biomérieux INSA INSA
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