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

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[[File:T--Aix-Marseille--EPSd.png|450px|right|thumb|EPS Depolymerase process to clean the xylem vessel from EPS.]]
 
[[File:T--Aix-Marseille--EPSd.png|450px|right|thumb|EPS Depolymerase process to clean the xylem vessel from EPS.]]
  
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To fight the problem that is [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']] we started searching for natural solutions against this bacterium and surely, we found bacteriophages. Many phages have a devious way to infect the bacterium; by cleaving the sugar bond in the biofilm they found more easily their target <ref>Vandenbergh, P. A., Wright, A. M. & Vidaver, A. K. Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora. Appl. Environ. Microbiol. 49, 994–996 (1985)</ref>.
+
To fight [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']] we searched for natural solutions.
 +
Some phages have a devious way to attack bacteria, digesting the biofilm so that they can their target more easily <ref>Vandenbergh, P. A., Wright, A. M. & Vidaver, A. K. Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora. Appl. Environ. Microbiol. 49, 994–996 (1985)</ref>.
 +
We decided to try to use this approach to attack [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']].
 +
We found an enzyme called EPS-depolymerase <ref>Kim, W. S. & Geider, K. Characterization of a Viral EPS-Depolymerase, a Potential Tool for Control of Fire Blight. Phytopathology 90, 1263–1268 (2000).</ref> that can hydrolyze the Exopolysaccharide (EPS) forming the biofilm.
  
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As the symptoms observed in plants are the result of water stress resulting from occlusion of xylem vessels by bacterial biofilm and/or accumulation of extracellular polysaccharides, we wanted to find a way to free the xylem vessels. The goal of this design isn’t to treat [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] or to prevent the infection, but to usen a enzyme called EPS-depolymerase <ref>Kim, W. S. & Geider, K. Characterization of a Viral EPS-Depolymerase, a Potential Tool for Control of Fire Blight. Phytopathology 90, 1263–1268 (2000).</ref>.
+
As the symptoms observed in plants are the result occlusion of xylem vessels by bacterial biofilm and the accumulation of EPS,  
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+
we think this may be a way to unblock the vessels.  
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This enzyme has the capacity to hydrolyze polysaccharidic bonds of the EPS composing the biofilm, which can free the xylem vessels.
+
The goal of part of the design isn’t to treat [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] or to prevent the infection, but to reduce symptoms.
  
 
==EPS Depolymerase design==
 
==EPS Depolymerase design==
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[[File:T--Aix-Marseille--EPSplasmid-2.png|450px|right|thumb]]
 
[[File:T--Aix-Marseille--EPSplasmid-2.png|450px|right|thumb]]
  
−
As we wanted to limit the number of GMOs in our product we wanted a production of the enzyme in ''E. coli'' and an efficient purification system.  
+
As we wanted to limit the number of GMOs in our product decided to purify the enzyme after production in ''E. coli'' using a quick and efficient purification system.  
  
−
The original enzyme used by the phage is long and its genes are not made for a production in ''E. coli''. Thus, to produce large quantities of the enzyme, we thought about the optimization of the enzyme by only taking the functional part of the original enzyme and we optimized its sequence for a production in ''E. coli''. This lead us to the creation of the EPS-Depolymerase part : [http://parts.igem.org/Part:BBa_K2255006 BBa_K2255006].  
+
The original enzyme used by the phage is very large and the gene is not optimal for a production in ''E. coli''.  
 +
Thus, to produce large quantities of the enzyme, we decided to optimize the enzyme by taking only the catalytic domain of the original protein and optimizing the gene sequence for production in ''E. coli''.  
 +
This lead to the creation of the EPS-Depolymerase part : [http://parts.igem.org/Part:BBa_K2255006 BBa_K2255006].  
  
−
As we wanted to purify the EPS Depolymerase for its use in our cure, we also engineered a multi-tag. This tag is composed of a histidine-tag, that binds to Nickel or Cobalt ions, and a Strep-tag that binds to streptavidine. Thus we created the biobrick [http://parts.igem.org/Part:BBa_K2255003 BBa_K2255003].
+
As we wanted to purify the EPS Depolymerase to use it in our treatment, we also engineered a multi-tag.  
 +
This tag is composed of an oligo-histidine-tag, that binds to Nickel or Cobalt ions, and a Strep-tag that binds to streptavidin separated by a TEV cleavage site.  
 +
Thus we created the biobrick [http://parts.igem.org/Part:BBa_K2255003 BBa_K2255003].
  
−
Those biobricks will be fused with the help of the [http://parts.igem.org/Assembly_standard_25 Rfc25] restriction site. Finally, to produce the enzyme in huge amounts, we decided to add a strong and constitutive promoter in ''E.coli'' ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]).
+
The two biobricks will be fused using the [http://parts.igem.org/Assembly_standard_25 Rfc25] standard to produce an easilly purifiable enzyme.
 +
To get large amounts, we decided to add a strong and constitutive promoter and RBS in ''E.coli'' ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]).
  
 
==References==
 
==References==
  
 
<references/>
 
<references/>

Revision as of 11:55, 1 November 2017

EPS Depolymerase

EPS Depolymerase process to clean the xylem vessel from EPS.

To fight Xylella fastidiosa we searched for natural solutions. Some phages have a devious way to attack bacteria, digesting the biofilm so that they can their target more easily [1]. We decided to try to use this approach to attack Xylella fastidiosa. We found an enzyme called EPS-depolymerase [2] that can hydrolyze the Exopolysaccharide (EPS) forming the biofilm.

As the symptoms observed in plants are the result occlusion of xylem vessels by bacterial biofilm and the accumulation of EPS, we think this may be a way to unblock the vessels. The goal of part of the design isn’t to treat X. fastidiosa or to prevent the infection, but to reduce symptoms.

EPS Depolymerase design

T--Aix-Marseille--EPSplasmid-2.png

As we wanted to limit the number of GMOs in our product decided to purify the enzyme after production in E. coli using a quick and efficient purification system.

The original enzyme used by the phage is very large and the gene is not optimal for a production in E. coli. Thus, to produce large quantities of the enzyme, we decided to optimize the enzyme by taking only the catalytic domain of the original protein and optimizing the gene sequence for production in E. coli. This lead to the creation of the EPS-Depolymerase part : [http://parts.igem.org/Part:BBa_K2255006 BBa_K2255006].

As we wanted to purify the EPS Depolymerase to use it in our treatment, we also engineered a multi-tag. This tag is composed of an oligo-histidine-tag, that binds to Nickel or Cobalt ions, and a Strep-tag that binds to streptavidin separated by a TEV cleavage site. Thus we created the biobrick [http://parts.igem.org/Part:BBa_K2255003 BBa_K2255003].

The two biobricks will be fused using the [http://parts.igem.org/Assembly_standard_25 Rfc25] standard to produce an easilly purifiable enzyme. To get large amounts, we decided to add a strong and constitutive promoter and RBS in E.coli ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]).

References

  1. ↑ Vandenbergh, P. A., Wright, A. M. & Vidaver, A. K. Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora. Appl. Environ. Microbiol. 49, 994–996 (1985)
  2. ↑ Kim, W. S. & Geider, K. Characterization of a Viral EPS-Depolymerase, a Potential Tool for Control of Fire Blight. Phytopathology 90, 1263–1268 (2000).