Team:Paris Saclay/Project

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{{Team:Paris_Saclay/incl_menu_navigation}}
{{Team:Paris_Saclay/incl_menu_navigation}}
Project
Project
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[[Team:Paris_Saclay/Project|Description]]
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[[Team:Paris_Saclay/PCBs|What are PCBs ?]]
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[[Team:Paris_Saclay/Parts|Biobricks]]
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[[Team:Paris_Saclay/Project|Overview]]
[[Team:Paris_Saclay/Modeling|Modeling]]
[[Team:Paris_Saclay/Modeling|Modeling]]
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[[Team:Paris_Saclay/Safety|Safety]]
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[[Team:Paris_Saclay/PS-PCR|PS-PCR]]
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[[Team:Paris_Saclay/Achievements|Achievements]]
{{Team:Paris_Saclay/incl_fin_menu_navigation}}
{{Team:Paris_Saclay/incl_fin_menu_navigation}}
{{Team:Paris_Saclay/incl_contenu}}
{{Team:Paris_Saclay/incl_contenu}}
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<big>
 
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= '''What are PCBs?''' =
 
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<br>PolyChlorinated Biphenyls (PCBs), are a family of man-made aromatic organic chemicals which consist of a phenyl ring and more or less chlorines. The first PCB was synthesized in 1881 and were developed in the 1940’s. The production of PCBs peaked in 1970’s, but after discovering that PCBs harm human health, their the production of PCBs has been banned in 1979 in U.S. [[File:cautionpcb.jpg|left|250px]]And in France, the total interdict of production and usage of PCBs declared valid in 1987. The Stockholm Convention on Persistent Organic Pollutants (POPs) was signed in 2001, which aims to eliminate or restrict the production and usage of PCBs and other persistent organic pollutants. This world-wide ban of PCBs effective from May 2004.<br>
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= '''Detection and degradation of PCB system in ''Escherichia coli''''' =
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Nowadays, although the manufacture of PCBs ceased many years ago, because of PCBs’ high stability they are still dangerous for both the environment and human health. This is the reason why we want to accelerate the processes of Biodegradation of PCBs by using Biosynthetic method.
 
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Since the second half of the 20th century, scientists are fully aware that some bacterial species living in media with high concentration of PCBs are able to degrade PCBs into pyruvate and acetyl-CoA which are then easily metabolized by these organisms.
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These bacterial species structure in biofilm with regions that have variable concentrations of oxygen, high at the surface and decreasing with depth. Bacteria living in this habitat have, in most cases, different degradation pathways, which are aerobic or anaerobic depending on their spatial disposition in the biofilm.
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<br>
 
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= '''Chronology of PCBs''' =
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Bacteria in aerobic environment use PCB oxidative degradation pathways; those in anaerobic condition degrade PCBs via reductive dechlorination pathways. None of the bacteria seems to use both pathways.
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<br>
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{| class="chronologypcb" cellpadding="2" border="1" align="center"
 
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! scope="col"width="20"|YEAR
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The reductive dechlorination reduces the number of chlorines of high chlorinated PCBs. The dechlorinated PCBs can be further degraded by an oxidative degradation which is efficient only with low chlorinated PCBs. That’s may explain why these different species coexist in biofilms.
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! scope="col"width="40"|EVENTS
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|-
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|1881||align="center"|First PCBs synthesized
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|-
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|1927||align="center"|PCBs were first manufactured commercially
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|-
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|1968||align="center"|Yusho disease in Japan
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|-
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|1973||align="center"|OCDE suggested of using PCBs in close system
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|-
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|1975||align="center"|Ban of PCBs for open-air application in France
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|-
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|1979||align="center"|Ban of PCBs production in U.S
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|-
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|1987||align="center"|Ban of PCBs production and usage in France
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|-
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|1992||align="center"|OSPAR COMMISSION, made decision of abandonment of PCBs
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|-
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|2001||align="center"|The Stockholm Convention on Persistent Organic Pollutants signed
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|}
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<br>
 
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'''Our goal in this project is to desing an organism able to i) detect PCB and then ii)
 +
'''employ a sequential degradation of the PCB using both combined pathways.'''
 +
'''For our experiences, we used bacteria present in nature that are able to detect and degrade the'''
 +
'''PCBs, namely [http://en.wikipedia.org/wiki/Burkholderia_xenovorans ''Burkholderia xenovorans''], [http://www.ncbi.nlm.nih.gov/pubmed/22843571''Pseudomonas pseudoalcaligenes'' KF 707] and
 +
[http://en.wikipedia.org/wiki/Rhodococcus'''''Rhodococcus jostii'' RHA1].'''''
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= '''Why are PCBs persistent in the environment?''' =
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[[File:Psnotenough.png|center|200px]]
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<br>
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[[File:molecularpcb.jpg|thumb]]
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==Construction of a system to detect the presence of PCBs==
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PCBs are chlorinated derivative of biphenyls which are distinguished by the number and the position of chlorines. Because of those substituted chlorines, the degradation of PCBs releases Cl- which is toxic for almost all organisms. Organisms should be capable to deal with these Cl- ion during degrading PCBs. However, some microbes are able to decompose PCBs by two series of processes: replacement of chlorine by hydrogen known as reductive dechlorination; then use these biphenyl or as a carbon source. But in a nature system, the degradation of PCBs needs a microbial community to eliminate all PCBs, these processes are extremely slow. So in our project, we try to combine these 2 processes in one cell of bacterial.<br>
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''Pseudomonas pseudoalcaligenes'' expresses enzymes that are responsible for an oxidative degradation of PCBs. The system is regulated two proteins, BphR2 and BphR1 coded by the ''bphR2'' and ''bphR1'' genes, respectively. PCBs induce a BphR2 conformational change to trigger BphR2 transcriptional activity leading to expression of the PCB oxidative degradation pathway.
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[[File:colorpcb.jpg|left|180px]]There are up to 209 individual chlorinated compounds in total. They are chemically fairly inert, being extremely resistant to oxidation, reduction, addition, elimination, and electrophilic substitution reactions except under extreme conditions. According to a guideline of U.S EPA, the incineration for eliminating PCBs demands a temperature over 1200 degree Celsius in order to destroy totally PCBs and prevent some by-product such like dioxin.<br>
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[[File:PsR2degradation.jpg]]
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PCBs are either oily liquids or solids that are colorless to light yellow, have low solubility in water but highly soluble in organic solvents. This high lipophilicity causes PCBs to stay in animal fats and accumulate in food chain.
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BphR2 also induces the expression of ''bphR1''.
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<br>
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[[File:PsR2surR1.jpg]]
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= '''Detection and degradation of PCB system in E.coli''' =
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For the project, we will use the ''bphR2'' gene and the ''bphR1'' promoter. We will place the ''bphR2'' coding sequence under a constitutive promoter. We will also construct a transcriptional fusion between the ''bphR1'' promoter with the ''lacZ'' gene coding for the β-galactosidase enzyme. The amount of β-galactosidase can be easily monitored by a chemical reaction using Xgal. With this system, the β-galactosidase activity will dependent on the ''bphR1'' promoter expression. Since the ''bphR1'' promoter is controlled by the PCB-activated BphR2, the β-galactosidase activity will correlate with the presence of PCBs.
 +
[[File:PsavecPCB.jpg|800px]]
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Since the second half of the XXth century scientists are fully aware of the fact that some
+
==Combination of the aerobic and anaerobic PCB degradation pathways==
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species of bacteria living in mediums with high concentrations of PCB are able to degrade the
+
-
PCB in pyruvate and acetyl CoA easily metabolized by these organisms.
+
 +
To perform an efficient PCB degradation, two processes should be sequentially combined, the PCB reductive dechlorination followed by a PCB oxidative degradation pathway. Our goal is to engineer a bacterium expressing alternatively both pathways according to growth conditions, with first the reductive dechlorination in anaerobiosis followed by a PCB oxidative degradation in aerobiosis.
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These species structure biofilms were there are regions with variables concentration of
+
The bacterium ''E. coli'' has an aerobic and an anaerobic metabolism that will be used to combine of the two PCB degradation pathways. The switch between aerobic and anaerobic metabolism is partly regulated the transcriptional regulator FNR. This protein has a dual function: it activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism. FNR expression is constitutive, but its activity is directly affected by the presence of oxygen which oxidizes of an essential [4Fe-4S] cluster. For the project, we use two promoters, PnrdH and PnifR which are repressed and activated by FNR, respectively
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oxygen, decreasing with depth to surface. The bacteria living in this habitat have in most of
+
-
case different degradation pathways namely aerobic or anaerobic depending on the spatial
+
-
disposition in the biofilm.
+
 +
The reductive dechlorination pathway is not well characterized; only one enzyme, a dehalogenase, is mentioned as contributing to this pathway. We propose to clone the corresponding gene in ''E. coli'' under the control of the PnifR promoter. As a result, the reductive dechlorination enzyme should be expressed in anaerobiosis to perform the first PCB degradation step. The second step involves an oxidative degradation and is performed in aerobiosis. We propose to clone the operon PCB oxidative degradation under the PrndH promoter which is derepressed in anaerobiosis. This configuration should optimize the PCB degradation according to the environmental conditions.
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Bacteria in aerobic conditions use an aerobic degradation pathway, the PCB oxidative
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[[File:Psbigschema.jpg|800px]]
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degradation, and these in anaerobic condition the PCB reductive dechlorination; no one can
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use both pathways so as to degrade the PCB.
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 +
==References==
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The reductive dechlorination can reduce the number of chlorines in high chlorinated PCB
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Kensuke Furukawa, Hikaru Suenaga and Masatoshi Goto
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making them assimilable by the oxidative degradation, only efficient with low chlorinated
+
-
PCB. That’s the reason why these different species coexist in the biofilms.
+
 +
Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution
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'''So our goal in this project is the creation of an organism able to first detect PCB and after
+
JOUNAL OF BACTERIOLOGY, 2004
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'''employ a sequential degradation of the PCB using both combined pathways.'''
+
-
'''For our experiences we used bacteria present in nature that are able to detect and degrade the'''
+
-
'''PCB namely ''Burkholderia xenovorans, Pseudomonas pseudoalcaligenes KF 707,
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'''Rhodococcus jostii RHA1''.''''''
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[[File:Psnotenough.png|center|200px]]
 
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Kazunari Taira, Jun Hirose, Shinsaku Hayashida, and Kensuke Furukawa
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==Detection and report of the PCB==
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Analysis of bph Operon from the Polychlorynated Biphenyl-degrading Strain of ''Pseudomonas pseudoalcaligenes'' KF707
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In nature these bacteria have a system for the regulation of the oxidative degradation of PCB.
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THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1992
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This one is based on two regulatory proteins namely Bphr2 and Bphr1 coded respectively by
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the genes of the same name bphr2 and bphr1.
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Bphr2 is able to detect PCB that induces a modification of the protein conformation activating
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Kensuke Furukawa and Hidehiko Fujihara
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the beginning of the gene cluster coding for the enzymes doing the oxidative degradation but
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also the gene coding for the Bphr1 protein.
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Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features
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The Bphr1 protein can detect the HO-PCB a metabolite derived from PCB, a product of the
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JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2008
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beginning of oxidation reactions. In presence of OH-PCB it induces his own transcription and
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also the following genes from the cluster that will completely degrade the PCB.
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For our construct we will pick out the bphr2 gene and the promoter of the bphr1 gene induced
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by PCB-Bphr2 from our species. We will combine the bphr2 coding sequence with a
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constitutive promoter that makes up the detection system and finally we will combine the
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Bphr1 promoter with the lacZ gene coding for the β-galactosidase enzyme so as to do a
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chemical dosing with Xgal and report the signal.
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 +
Jim A. Field, Reyes Sierra-Alvarez
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[[File:Psdegradationexplication.jpg|center]]
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Microbial transformation and degradation of polychlorinated biphenyls
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-
 
+
-
 
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==Combination of the aerobic and anaerobic PCB degradation pathways==
+
-
 
+
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The bacteria E.coli has an aerobic and an anaerobic metabolism that’s why we used it for the
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Environmental Pollution, 2008
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combination of the two degradation pathways. The regulation between pathways in these two
+
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conditions is normally made by regulatory proteins like FNR.
+
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The FNR protein modifies its conformation in presence of oxygen having an activator or an
+
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inhibitor function.
+
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The reductive dechlorination pathway is not well characterized only an enzyme, a
+
Lorenz Adrian, Helmut Görisch
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dehalogenase, is mentioned as contributing to this pathway. In these anaerobic conditions the
+
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chlorine takes the place of the oxygen as the electron acceptor.
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That’s why we have chosen an activator FNR in presence of oxygen in order to activate the
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oxidative degradation.
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Microbial transformation of chlorinated benzenes under anaerobic conditions
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Research in Microbiology, 2002
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[[File:Psdegradationexplication2.jpg|center|800px]]
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Dean. A Tolla and Michael A. Savageau
 +
Regulation of Aerobic-to-Anaerobic Transitions by the FNR Cycle in Escherichia coli
 +
J. Mol. Biol. (2010)
 +
Hidehiko Fujihara, Hideyuki Yoshida, Tetsuya Matsunaga, Masatoshi Goto, and Kensuke Furukawa
 +
Cross-Regulation of Biphenyl- and Salicylate-Catabolic Genes by Two Regulatory Systems in Pseudomonas pseudoalcaligenes KF707
 +
JOURNAL OF BACTERIOLOGY, July 2006
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Team project descriptions are due <strong>August 9</strong>.  The description is only a preliminary description - it will not be used to judge your project.  What you write will only serve to provide some background on what your team has been working on so far and what you hope to accomplish. 
 
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<strong>Description requirements:</strong>
 
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* Describe your project on the front page of your team's wiki* or on another page that is easily reached. 
 
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* The description only needs to be a couple of paragraphs long.
 
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'''*Note:''' The project description does not need to be emailed to iGEM HQ.
 
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</big>
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Article written by Eric and Nadia
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 21:50, 4 October 2013

Project What are PCBs ? Overview Modeling PS-PCR Achievements

Contents

Detection and degradation of PCB system in Escherichia coli

Since the second half of the 20th century, scientists are fully aware that some bacterial species living in media with high concentration of PCBs are able to degrade PCBs into pyruvate and acetyl-CoA which are then easily metabolized by these organisms.


These bacterial species structure in biofilm with regions that have variable concentrations of oxygen, high at the surface and decreasing with depth. Bacteria living in this habitat have, in most cases, different degradation pathways, which are aerobic or anaerobic depending on their spatial disposition in the biofilm.


Bacteria in aerobic environment use PCB oxidative degradation pathways; those in anaerobic condition degrade PCBs via reductive dechlorination pathways. None of the bacteria seems to use both pathways.


The reductive dechlorination reduces the number of chlorines of high chlorinated PCBs. The dechlorinated PCBs can be further degraded by an oxidative degradation which is efficient only with low chlorinated PCBs. That’s may explain why these different species coexist in biofilms.


Our goal in this project is to desing an organism able to i) detect PCB and then ii) employ a sequential degradation of the PCB using both combined pathways. For our experiences, we used bacteria present in nature that are able to detect and degrade the PCBs, namely [http://en.wikipedia.org/wiki/Burkholderia_xenovorans Burkholderia xenovorans], [http://www.ncbi.nlm.nih.gov/pubmed/22843571Pseudomonas pseudoalcaligenes KF 707] and [http://en.wikipedia.org/wiki/RhodococcusRhodococcus jostii RHA1].

Psnotenough.png


Construction of a system to detect the presence of PCBs

Pseudomonas pseudoalcaligenes expresses enzymes that are responsible for an oxidative degradation of PCBs. The system is regulated two proteins, BphR2 and BphR1 coded by the bphR2 and bphR1 genes, respectively. PCBs induce a BphR2 conformational change to trigger BphR2 transcriptional activity leading to expression of the PCB oxidative degradation pathway.

PsR2degradation.jpg

BphR2 also induces the expression of bphR1.

PsR2surR1.jpg

For the project, we will use the bphR2 gene and the bphR1 promoter. We will place the bphR2 coding sequence under a constitutive promoter. We will also construct a transcriptional fusion between the bphR1 promoter with the lacZ gene coding for the β-galactosidase enzyme. The amount of β-galactosidase can be easily monitored by a chemical reaction using Xgal. With this system, the β-galactosidase activity will dependent on the bphR1 promoter expression. Since the bphR1 promoter is controlled by the PCB-activated BphR2, the β-galactosidase activity will correlate with the presence of PCBs.

PsavecPCB.jpg

Combination of the aerobic and anaerobic PCB degradation pathways

To perform an efficient PCB degradation, two processes should be sequentially combined, the PCB reductive dechlorination followed by a PCB oxidative degradation pathway. Our goal is to engineer a bacterium expressing alternatively both pathways according to growth conditions, with first the reductive dechlorination in anaerobiosis followed by a PCB oxidative degradation in aerobiosis.

The bacterium E. coli has an aerobic and an anaerobic metabolism that will be used to combine of the two PCB degradation pathways. The switch between aerobic and anaerobic metabolism is partly regulated the transcriptional regulator FNR. This protein has a dual function: it activates genes involved in anaerobic metabolism and represses genes involved in aerobic metabolism. FNR expression is constitutive, but its activity is directly affected by the presence of oxygen which oxidizes of an essential [4Fe-4S] cluster. For the project, we use two promoters, PnrdH and PnifR which are repressed and activated by FNR, respectively

The reductive dechlorination pathway is not well characterized; only one enzyme, a dehalogenase, is mentioned as contributing to this pathway. We propose to clone the corresponding gene in E. coli under the control of the PnifR promoter. As a result, the reductive dechlorination enzyme should be expressed in anaerobiosis to perform the first PCB degradation step. The second step involves an oxidative degradation and is performed in aerobiosis. We propose to clone the operon PCB oxidative degradation under the PrndH promoter which is derepressed in anaerobiosis. This configuration should optimize the PCB degradation according to the environmental conditions.

Psbigschema.jpg

References

Kensuke Furukawa, Hikaru Suenaga and Masatoshi Goto

Biphenyl Dioxygenases: Functional Versatilities and Directed Evolution

JOUNAL OF BACTERIOLOGY, 2004


Kazunari Taira, Jun Hirose, Shinsaku Hayashida, and Kensuke Furukawa

Analysis of bph Operon from the Polychlorynated Biphenyl-degrading Strain of Pseudomonas pseudoalcaligenes KF707

THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1992


Kensuke Furukawa and Hidehiko Fujihara

Microbial Degradation of Polychlorinated Biphenyls: Biochemical and Molecular Features

JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2008


Jim A. Field, Reyes Sierra-Alvarez

Microbial transformation and degradation of polychlorinated biphenyls

Environmental Pollution, 2008


Lorenz Adrian, Helmut Görisch

Microbial transformation of chlorinated benzenes under anaerobic conditions

Research in Microbiology, 2002


Dean. A Tolla and Michael A. Savageau

Regulation of Aerobic-to-Anaerobic Transitions by the FNR Cycle in Escherichia coli

J. Mol. Biol. (2010)


Hidehiko Fujihara, Hideyuki Yoshida, Tetsuya Matsunaga, Masatoshi Goto, and Kensuke Furukawa

Cross-Regulation of Biphenyl- and Salicylate-Catabolic Genes by Two Regulatory Systems in Pseudomonas pseudoalcaligenes KF707

JOURNAL OF BACTERIOLOGY, July 2006




Article written by Eric and Nadia

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