Difference between revisions of "Team:Aix-Marseille/QS"

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==Quorum Sensing Approach==
 
==Quorum Sensing Approach==
  
The quorum sensing is a mechanism involving chemical compounds that create a line of communication for various bacterium which regulate their behavior in the confined of their population. ''Xylella fastidiosa'' communicates by quorum sensing to coordinate the biofilm production and bacterial virulence with fatty acids. But, bacteria use  a set of instruction called the quorum quenching to blur the quorum sensing of other bacterial population.
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Quorum sensing is a mechanism that allows bacteria to coordinate the regulation of their genes according to the density of the bacterial population. It allows bacteria to adopt collective patterns of gene regulation to have, at a population level, an advantageous phenotype. Quorum sensing allows [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']] to produce a biofilm, a sticky extracellular matrix composed of DNA, proteins and polysaccharides, which is one of the key problem in the plant disease because it blocks xylem vessels.  
  
[[Team:Aix-Marseille/Quorum Sensing Approach|Read more…]]
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In order to produce this biofilm, to communicate and coordinate the gene expression of the whole colony, [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] mainly uses Diffusible Signal Factor (DSF) which are fatty acids with an insaturation in 2cis position. The same route of communication also regulates virulence. In addition to quorum sensing, bacteria can also use a similar mechanism called quorum quenching to blur the quorum sensing of other bacterial populations and outcompete them. Therefor to stop [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] to form the biofilm that causes the death of plants we choose to quench it quorum sensing activity.
  
==The Biobricks==
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''Pseudomonas aeruginosa'' under stress produces a specific fatty acid named 2-cis-decenoic acid, that has the ability to stop the biofilm production of many organisms. Thus, we thought about using this specific DSF to stop [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']].
  
According to the paper "Promiscuous Diffusible Signal Factor Production and Responsiveness of the Xylella fastidiosa Rpf System" and “The Putative Enoyl-Coenzyme A Hydratase DspI Is Required for Production of the Pseudomonas aeruginosa Biofilm Dispersion Autoinducer cis-Decenoic Acid”, we found the 2-cis-decenoic acid is produced by ''Pseudomonas aeruginosa'' under stress to degrade the biofilm of other organism. Including the biofilm of ''Xylella fastidiosa'', indeed the 2-cis-decanoic acid seems to inhibit the induction of genes depending on the quorum sensing of ''Xylella fastidiosa'' and therefor its biofilm.
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==Design==
Again in according to the paper “The Putative Enoyl-Coenzyme A Hydratase DspI Is Required for Production of the Pseudomonas aeruginosa Biofilm Dispersion Autoinducer cis-Decenoic Acid”, the enzyme DspI is necessary to the production of 2-cis-decenenoic acid by Pseudomonas aeruginosa, so we search the DNA sequence of the enzyme DspI in the genome of Pseudomonas aeruginosa to create our first biobrick ( part ).
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As we wanted to limit the number of GMOs in our product we wanted to produce the 2-cis-decenoic acid in ''E. coli'' and an efficient purification system. We choose to not use ''P. aeruginosa'' because of it pathogeneicity.
  
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Several enzymes are necessary for the production of 2-cis-decenenoic acid by ''Pseudomonas aeruginosa'', not every of them is present in ''E. coli''. Thus, to produce the fatty acid, we  design the three enzymes lacking in ''E. coli''  for the production of 2-cis-decenenoic and we optimized their sequences for a production in this bacteria. Therefore we design : [http://parts.igem.org/Part:BBa_K2255000 BBa_K2255000], [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002]
  
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As we wanted to optimize the production we design these parts to form a operon when assembled. Thus, [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002] has a Ribosome Binding Site (RBS) integrated in the biobrick.
  
''Cis-2-decenoic acid effect (DA) on biofilm production'''
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Finally, to produce these enzymes in huge amounts, we decided to add a strong and constitutive promoter in E.coli (BBa_K608002).
 
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According to the paper “The Putative Enoyl-Coenzyme A Hydratase DspI Is Required for Production of the Pseudomonas aeruginosa Biofilm Dispersion Autoinducer cis-Decenoic Acid”, biofilm dispersion assays are performed by addition of cis-2-decenoic (DA) acid with a concentration of 310 nM.
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We tested the effect of this fatty acid on Xanthomonas and Pseudomonas in differents parameters:fatty acid concentration, medium for bacteria, type of tube. The point of theses experiments are to show the effect of this molecule on biofilm production when we add it in medium at different moments of growth. To charaterize the quantity of biofilm, we use purple crystal coloration, that mark on tube a ring of biofilm. Then we mesure with TECAN the quantity of biofilm.
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'''Mesure OD600nm plaque bottom TECAN'''
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[[File:T--Aix-Marseille--plaqueviolettecan.jpeg|thumb|]]
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Revision as of 13:14, 19 October 2017

Quorum sensing


Quorum Sensing Approach

Quorum sensing is a mechanism that allows bacteria to coordinate the regulation of their genes according to the density of the bacterial population. It allows bacteria to adopt collective patterns of gene regulation to have, at a population level, an advantageous phenotype. Quorum sensing allows Xylella fastidiosa to produce a biofilm, a sticky extracellular matrix composed of DNA, proteins and polysaccharides, which is one of the key problem in the plant disease because it blocks xylem vessels.

In order to produce this biofilm, to communicate and coordinate the gene expression of the whole colony, X. fastidiosa mainly uses Diffusible Signal Factor (DSF) which are fatty acids with an insaturation in 2cis position. The same route of communication also regulates virulence. In addition to quorum sensing, bacteria can also use a similar mechanism called quorum quenching to blur the quorum sensing of other bacterial populations and outcompete them. Therefor to stop X. fastidiosa to form the biofilm that causes the death of plants we choose to quench it quorum sensing activity.

Pseudomonas aeruginosa under stress produces a specific fatty acid named 2-cis-decenoic acid, that has the ability to stop the biofilm production of many organisms. Thus, we thought about using this specific DSF to stop X. fastidiosa.

Design

As we wanted to limit the number of GMOs in our product we wanted to produce the 2-cis-decenoic acid in E. coli and an efficient purification system. We choose to not use P. aeruginosa because of it pathogeneicity.

Several enzymes are necessary for the production of 2-cis-decenenoic acid by Pseudomonas aeruginosa, not every of them is present in E. coli. Thus, to produce the fatty acid, we design the three enzymes lacking in E. coli for the production of 2-cis-decenenoic and we optimized their sequences for a production in this bacteria. Therefore we design : [http://parts.igem.org/Part:BBa_K2255000 BBa_K2255000], [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002]

As we wanted to optimize the production we design these parts to form a operon when assembled. Thus, [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002] has a Ribosome Binding Site (RBS) integrated in the biobrick.

Finally, to produce these enzymes in huge amounts, we decided to add a strong and constitutive promoter in E.coli (BBa_K608002).