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

 
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{{Aix-Marseille|title=EPS Depolymerase|toc=__TOC__}}
 
{{Aix-Marseille|title=EPS Depolymerase|toc=__TOC__}}
  
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To fight the problem that is [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']] we firstly started searching for natural solutions against this bacterium and surely, we found bacteriophages that loved destroying this bacterium. Many phages have a devious way to infect the bacterium; by cleaving the sugar bond in the biofilm and then adsorbing on the bacterial membrane, it sets off a series of events leading to the injection of the bacteriophage genome in the bacterium, triggering the life cycle of the phage.
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[[File:T--Aix-Marseille--EPSd.png|450px|right|thumb|EPS Depolymerase process to clean the xylem vessel from EPS.]]
  
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[[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] is a Gram-negative bacterium from the gammaproteobacteria) familie. 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, and the subsequent blockage of xylem vessels with pectins, tyloses and gums produced by the plant host in response to [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] infection.
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To fight [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']] we searched for natural solutions.
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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>.
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We decided to try to use this approach to attack [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']].
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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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One strategy was put in place to manage this serious pathogen and that is to inject the host with bacteriophages which are a class of extremely specific viruses that infect bacteria and lack the ability to infect more complex organisms such as mammals. Using phage will limit the long term effect to the soil or the microflora even to the animals that consume the fruits. The goal of this design isn’t to threat [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] or to prevent the infection, but to use a enzyme called EPS-depolymerase.
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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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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.
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==EPS Depolymerase design==
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[[File:T--Aix-Marseille--EPSplasmid-2.png|450px|right|thumb]]
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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.
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The original enzyme used by the phage is very large and the gene is not optimal for a production in ''E. coli''.
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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''.
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This lead to the creation of the EPS-Depolymerase part : [http://parts.igem.org/Part:BBa_K2255006 BBa_K2255006].
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As we wanted to purify the EPS Depolymerase to use it in our treatment, we also engineered a multi-tag.
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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.
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Thus we created the biobrick [http://parts.igem.org/Part:BBa_K2255003 BBa_K2255003].
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The two biobricks will be fused using the [http://parts.igem.org/Assembly_standard_25 Rfc25] standard to produce an easilly purifiable enzyme.
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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]).
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==References==
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<references/>

Latest revision as of 02:16, 2 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).