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

 
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{{Aix-Marseille|title=Quorum sensing|toc=__TOC__}}
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{{Aix-Marseille|title=Quorum sensing|toc=__noTOC__}}
  
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Quorum sensing is a mechanism that allows bacteria to coordinate their behaviour depending on the bacterial population density <ref>Rutherford, S. T. & Bassler, B. L. Bacterial Quorum Sensing: Its Role in Virulence and Possibilities for Its Control. Cold Spring Harb Perspect Med 2, a012427 (2012).</ref>.
 +
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 problems in the plant disease because it blocks xylem vessels.
  
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==Quorum Sening Approach==
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[[File:T--Aix-Marseille--QSexp.png|500px|right|thumb| Utilization of the DSF 2-cis-decenoic acid to stop the biofilm formation of bacteria.]]
  
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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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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 a Diffusible Signal Factor (DSF) which is a fatty acid with a 2-cis unsaturation<ref>Ionescu, M. et al. Promiscuous Diffusible Signal Factor Production and Responsiveness of the ''Xylella fastidiosa'' Rpf System. mBio 7, e01054–16 (2016).</ref>.
 +
The same system of communication regulates virulence.  
 +
In addition to quorum sensing, bacteria can also use a similar mechanism called quorum quenching to modify the quorum sensing of other bacterial populations and outcompete them.
 +
Therefore, to stop [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] forming the biofilm that kills plants we choose to quench it quorum sensing activity.  
  
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[[Team:Aix-Marseille/Quorum Sening Approach|Read more…]]
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''Pseudomonas aeruginosa'' under stress produces a similar fatty acid named 2-cis-decenoic acid, that is able to stop biofilm formation by many organisms<ref>Amari, D. T., Marques, C. N. H. & Davies, D. G. The Putative Enoyl-Coenzyme A Hydratase DspI Is Required for Production of the ''Pseudomonas aeruginosa'' Biofilm Dispersion Autoinducer cis-2-Decenoic Acid. J. Bacteriol. 195, 4600–4610 (2013).</ref>.
 +
Thus, we thought of including this specific DSF in '''KILL XYL''' to stop [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] biofilm formation.
  
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==The Biobricks==
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==Design==
  
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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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[[File:T--Aix-Marseille--pQS2.png|450px|right|thumb]]
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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 ).  
+
  
 +
As we wanted to limit the number of GMOs in our product we wanted to produce and purify the 2-cis-decenoic acid in ''E. coli''.
 +
We choose to not use ''P. aeruginosa'' because of it pathogenicity.
  
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Several enzymes are necessary for the production of 2-cis-decenoic acid by ''Pseudomonas aeruginosa'', thay are not all present in ''E. coli''.
 +
Thus, to produce the fatty acid, we designed biobricks to produce the three enzymes lacking in ''E. coli'' for the production of 2-cis-decenenoic and we optimized their sequences for production in ''E. coli''.
 +
Part [http://parts.igem.org/Part:BBa_K2255000 BBa_K2255000] is an enoyl-CoA hydratase, [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] an acyl-CoA isomerase and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002] a thioesterase. 
  
 +
We want to optimize the production of the different proteins so we designed these parts so they can be assembled to form an operon. Thus, [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002] have a Ribosome Binding Site (RBS) integrated into the biobrick.
 +
To produce large amounts of these enzymes, we decided to add a strong and constitutive promoter in ''E.coli'' ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]).
  
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''Cis-2-decenoic acid effect (DA) on biofilm production'''
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==Results==
  
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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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[[File:T--Aix-Marseille--biofilmtubeacide.jpeg|thumb|left|Biofilm production without fatty acid after 24, 48, 72, and 96 hours.]]
 +
[[File:T--Aix-Marseille--biofilmtubeacideeffet.jpeg|thumb|left|Biofilm production with fatty acid 24, 48, 72, and 96 hours.]]
 +
As a proof of concept, we studied the effect of the fatty acid on ''X. campestris''.
 +
We used several different experimental conditions, such as the concentration of product and type of tube.
 +
After growing bacteria in media containing the product, we measured the effect of it on biofilm production using crystal violet visually ([https://2017.igem.org/Team:Aix-Marseille/Notebook#Biofilm_quantification Protocol]).
 +
Biofilm production can also be quantified with TECAN and is proportional with absorbance.
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We measured fatty acid effect by adding it at the start of growth.
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Then we observed biofilm production after 24, 48, 72, and 96 hours.  
  
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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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These results are very encouraging. We can see a significant decrease of biofilm production after 48, 72 and 96 hours.
 +
However, they need to be reproduced and the biofilm quantified and studied in more detail.
  
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'''Mesure of biofilm density by coloration by purple crystal then sonication'''
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<!-- This does not help understanding numbers with no explanation and according to the figure 24 hours is not long enough
  
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The plaque containing the peignes is used to color the ring of biofilm by purple cristal. After rincing, the ring will be off hook by sonication and the purple cristal resolubilised will be mesured at DO 570nm.  
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We decided to replicate the mesure and quantify the experiments numerically.
  
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Protocol
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Raw Data:  *** YOU SHOULD NOT PRESENT RAW DATA ***
  
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#Add a drop of CV in each well (pipette). I let colore 10 minutes at 30°C .
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All the measures were made by Tecan Infinite® 200 at a wavelenght of 570nm.
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#Blow the liquide
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#Rince twice with water MiliQ
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#Add 250µl physiologic water (0.9% NaCl) + ethanol 60%
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#Agitation auto during 2 minutes
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#Mesure OD570nm.
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'''Mesure OD600nm plaque bottom TECAN''' 
 
  
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#Take off the cap
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#Agitation auto during 2 minutes (amplitude 3mm orbitale)
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{| style="float:left"
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#Mesure OD600 above through the plaque (25 flashes).
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|- style="background-color:#808080;color:#fff;"
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#The OD600 is proportional to biofilm production.
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|| Hours
 +
| align=right| 6
 +
| align=right| 12
 +
| align=right| 24
 +
|-
 +
| Replicat1
 +
| 0,33360001
 +
| 0,44459999
 +
| 0,78009999
 +
|-
 +
| Replicat2
 +
| 0,3337
 +
| 0,48379999
 +
| 0,91189998
 +
|-
 +
| Replicat3
 +
| 0,57870001
 +
| 0,49349999
 +
| 0,82230002
 +
|-
 +
| Average
 +
| 0,41533334
 +
| 0,47396666
 +
| 0,8381
 +
|-
 +
| Ecart-type
 +
| 0,14147969
 +
| 0,02589061
 +
| 0,06730557
 +
|-
 +
|}
 +
 
 +
{| style="float:left"
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|- style="background-color:#808080;color:#fff;"
 +
|| Hours
 +
| align=right| 6
 +
| align=right| 12
 +
| align=right| 24
 +
|-
 +
| Replicat1
 +
| 0,32260001
 +
| 0,54210001
 +
| 1,16499996
 +
|-
 +
| Replicat2
 +
| 0,456
 +
| 0,50389999
 +
| 0,82029998
 +
|-
 +
| Replicat3
 +
| 0,50999999
 +
| 0,40290001
 +
| 0,80269998
 +
|-
 +
| Average
 +
| 0,42953333
 +
| 0,48296667
 +
| 0,92933331
 +
|-
 +
| Ecart-type
 +
| 0,0964627
 +
| 0,07192227
 +
| 0,20428294
 +
|-
 +
|}
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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The second experiment consist in adding the product not directly in the medium but after a certain time of growth. We choose to add the fatty acid in a culture of 24 hours. We observed the effect at time 6 hours and 24 hours later.
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 +
 
 +
 
 +
 
 +
{| style="float:left"
 +
|- style="background-color:#808080;color:#fff;;"
 +
|| Hours
 +
| align=right| 6
 +
| align=right| 24
 +
|-
 +
| Replicat1
 +
| 0,97390002
 +
| 2,08780003
 +
|-
 +
| Replicat2
 +
| 0,97680002
 +
| 1,68789995
 +
|-
 +
| Replicat3
 +
| 0,99870002
 +
| 1,73870003
 +
|-
 +
| Average
 +
| 0,98313336
 +
| 1,83813334
 +
|-
 +
| Ecart-type
 +
| 0,01355888
 +
| 0,21770451
 +
|-
 +
|}
 +
 
 +
{| style="float:left"
 +
|- style="background-color:#808080;color:#fff;"
 +
|| Hours
 +
| align=right| 6
 +
| align=right| 24
 +
|-
 +
| Replicat1
 +
| 0,96560001
 +
| 1,44819999
 +
|-
 +
| Replicat2
 +
| 1,00259995
 +
| 1,79219997
 +
|-
 +
| Replicat3
 +
| 0,9734
 +
| 1,30620003
 +
|-
 +
| Average
 +
| 0,98053332
 +
| 1,51553333
 +
|-
 +
| Ecart-type
 +
| 0,01950416
 +
| 0,24989862
 +
|-
 +
|}
 +
 
 +
<div style="clear:both"></div>
 +
 
 +
 
 +
 
 +
 
 +
Contrary to a first visual quantification, results are inconclusive. Actually, there is not a significant difference beetween standard conditions and the add of fatty acid.  
 +
However, we noted a small reduction when the fatty acid is used after a period of growth. This difference let us think that maybe the fatty acid have a stronger impact on the current production.
 +
Finally, visuals results bring to light some impact which deserve more research with different parameters.
 +
 
 +
-->
 +
 
 +
==References==

Latest revision as of 03:35, 2 November 2017

Quorum sensing

Quorum sensing is a mechanism that allows bacteria to coordinate their behaviour depending on the bacterial population density [1]. 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 problems in the plant disease because it blocks xylem vessels.

Utilization of the DSF 2-cis-decenoic acid to stop the biofilm formation of bacteria.

In order to produce this biofilm, to communicate and coordinate the gene expression of the whole colony, X. fastidiosa mainly uses a Diffusible Signal Factor (DSF) which is a fatty acid with a 2-cis unsaturation[2]. The same system of communication regulates virulence. In addition to quorum sensing, bacteria can also use a similar mechanism called quorum quenching to modify the quorum sensing of other bacterial populations and outcompete them. Therefore, to stop X. fastidiosa forming the biofilm that kills plants we choose to quench it quorum sensing activity.

Pseudomonas aeruginosa under stress produces a similar fatty acid named 2-cis-decenoic acid, that is able to stop biofilm formation by many organisms[3]. Thus, we thought of including this specific DSF in KILL XYL to stop X. fastidiosa biofilm formation.

Design

T--Aix-Marseille--pQS2.png

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

Several enzymes are necessary for the production of 2-cis-decenoic acid by Pseudomonas aeruginosa, thay are not all present in E. coli. Thus, to produce the fatty acid, we designed biobricks to produce the three enzymes lacking in E. coli for the production of 2-cis-decenenoic and we optimized their sequences for production in E. coli. Part [http://parts.igem.org/Part:BBa_K2255000 BBa_K2255000] is an enoyl-CoA hydratase, [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] an acyl-CoA isomerase and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002] a thioesterase.

We want to optimize the production of the different proteins so we designed these parts so they can be assembled to form an operon. Thus, [http://parts.igem.org/Part:BBa_K2255001 BBa_K2255001] and [http://parts.igem.org/Part:BBa_K2255002 BBa_K2255002] have a Ribosome Binding Site (RBS) integrated into the biobrick. To produce large amounts of these enzymes, we decided to add a strong and constitutive promoter in E.coli ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]).

Results

Biofilm production without fatty acid after 24, 48, 72, and 96 hours.
Biofilm production with fatty acid 24, 48, 72, and 96 hours.

As a proof of concept, we studied the effect of the fatty acid on X. campestris. We used several different experimental conditions, such as the concentration of product and type of tube. After growing bacteria in media containing the product, we measured the effect of it on biofilm production using crystal violet visually (Protocol). Biofilm production can also be quantified with TECAN and is proportional with absorbance. We measured fatty acid effect by adding it at the start of growth. Then we observed biofilm production after 24, 48, 72, and 96 hours.

These results are very encouraging. We can see a significant decrease of biofilm production after 48, 72 and 96 hours. However, they need to be reproduced and the biofilm quantified and studied in more detail.


References

  1. ↑ Rutherford, S. T. & Bassler, B. L. Bacterial Quorum Sensing: Its Role in Virulence and Possibilities for Its Control. Cold Spring Harb Perspect Med 2, a012427 (2012).
  2. ↑ Ionescu, M. et al. Promiscuous Diffusible Signal Factor Production and Responsiveness of the Xylella fastidiosa Rpf System. mBio 7, e01054–16 (2016).
  3. ↑ Amari, D. T., Marques, C. N. H. & Davies, D. G. The Putative Enoyl-Coenzyme A Hydratase DspI Is Required for Production of the Pseudomonas aeruginosa Biofilm Dispersion Autoinducer cis-2-Decenoic Acid. J. Bacteriol. 195, 4600–4610 (2013).