Difference between revisions of "Part:BBa K2043001"
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NOTOC__
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__NOTOC__
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<partinfo>BBa_K2043001 short</partinfo>
 
<partinfo>BBa_K2043001 short</partinfo>
  
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This is catechol-1,2-dioxygenase from Acinetobacter pittii codon optimized for E.coli with His-tag for easy purification
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This part corresponds to <b>Catechol-1,2-dioxygenase</b> cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from <i>Acinetobacter pittii</i>, which we <b>codon optimised for <i>E. coli</i></b>.<br>
 +
In order to facilitate working with this enzyme, we added a <b>His-tag</b> at the <b>C-terminal</b>. This tag allows for purification in an easier way.<br><br>
 +
<br>
 +
We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us. <br>
 +
In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.<br>
 +
 
 +
<b>Testing the part</b><br><br>
 +
 
 +
We tested the activity of CatA using cell extract of cells expressing our protein. <br>
 +
First, we performed an SDS-PAGE to check whether the protein was being expressed. <br>
 +
https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg
 +
<br><br>
 +
 
 +
The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity. <br>
 +
We tested our cell extract for CatA activity in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed. <br>
 +
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product. <br>
 +
https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg
 +
 
  
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
 
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<h1>catA - codon optimized</h1>
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<p>put info here :)</p>
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<h1>GFP-FBD2</h1>
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<pre>
 +
                          ........
 +
                          ;::;;::;,
 +
                          ;::;;::;;,
 +
                          ;;:::;;::;;,
 +
          .vnmmnv%vnmnv%,.;;;:::;;::;;,  .,vnmnv%vnmnv,
 +
      vnmmmnv%vnmmmnv%vnmmnv%;;;;;;;%nmmmnv%vnmmnv%vnmmnv
 +
    vnmmnv%vnmmmmmnv%vnmmmmmnv%;:;%nmmmmmmnv%vnmmmnv%vnmmmnv
 +
    vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmnv%vnmmmnv%vnmmmnv
 +
  vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmmmnv%vnmmmnv%vnmmmnv
 +
  vnmmnv%vnmmmmmnv%vnmm;mmmmmmnv%vnmmmmmmmm;mmnv%vnmmmnv%vnmmmnv,
 +
vnmmnv%vnmmmmmnv%vnmm;' mmmmmnv%vnmmmmmmm;' mmnv%vnmmmnv%vnmmmnv
 +
vnmmnv%vnmmmmmnv%vn;;    mmmmnv%vnmmmmmm;;    nv%vnmmmmnv%vnmmmnv
 +
vnmmnv%vnmmmmmmnv%v;;      mmmnv%vnmmmmm;;      v%vnmmmmmnv%vnmmmnv
 +
vnmmnv%vnmmmmmmnv%vnmmmmmmmmm;;      mmmmmmmmmnv%vnmmmmmmnv%vnmmmnv
 +
vnmmnv%vnmmmmmmnv%vnmmmmmmmmmm;;    mmmmmmmmmmnv%vnmmmmmmnv%vnmmmnv
 +
vnmmnv%vnmmmmm nv%vnmmmmmmmmmmnv;, mmmmmmmmmmmmnv%vn;mmmmmnv%vnmmmnv
 +
vnmmnv%vnmmmmm  nv%vnmmmmmmmmmnv%;nmmmmmmmmmmmnv%vn; mmmmmnv%vnmmmnv
 +
`vnmmnv%vnmmmm,  v%vnmmmmmmmmmmnv%vnmmmmmmmmmmnv%v;  mmmmnv%vnnmmnv'
 +
vnmmnv%vnmmmm;,  %vnmmmmmmmmmnv%vnmmmmmmmmmnv%;'  mmmnv%vnmmmmnv
 +
  vnmmnv%vnmmmm;;,  nmmm;'              mmmm;;'    mmmnv%vnmmmmnv'
 +
  `vnmmnv%vnmmmmm;;,.        mmnv%v;,            mmmmnv%vnmmmmnv'
 +
  `vnmmnv%vnmmmmmmnv%vnmmmmmmmmnv%vnmmmmmmnv%vnmmmmmnv%vnmmmmnv'
 +
    `vnmvn%vnmmmmmmnv%vnmmmmmmmnv%vnmmmmmnv%vnmmmmmnv%vnmmmnv'
 +
        `vn%vnmmmmmmn%:%vnmnmmmmnv%vnmmmnv%:%vnmmnv%vnmnv'
 +
 
 +
</pre>

Revision as of 17:09, 21 October 2016

NOTOC__ catA from Acinetobacter pittii, codon optimized for E. coli

This part corresponds to Catechol-1,2-dioxygenase cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from Acinetobacter pittii, which we codon optimised for E. coli.
In order to facilitate working with this enzyme, we added a His-tag at the C-terminal. This tag allows for purification in an easier way.


We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us.
In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.

Testing the part

We tested the activity of CatA using cell extract of cells expressing our protein.
First, we performed an SDS-PAGE to check whether the protein was being expressed.
Paris_Bettencourt_notebook_GELS.jpg

The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity.
We tested our cell extract for CatA activity in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.
Paris_Bettencourt_notebook_catA_good.jpg



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



GFP-FBD2

                          ........ 
                           ;::;;::;, 
                           ;::;;::;;, 
                          ;;:::;;::;;, 
          .vnmmnv%vnmnv%,.;;;:::;;::;;,  .,vnmnv%vnmnv, 
       vnmmmnv%vnmmmnv%vnmmnv%;;;;;;;%nmmmnv%vnmmnv%vnmmnv 
     vnmmnv%vnmmmmmnv%vnmmmmmnv%;:;%nmmmmmmnv%vnmmmnv%vnmmmnv 
    vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmnv%vnmmmnv%vnmmmnv 
   vnmmnv%vnmmmmmnv%vnmmmmmmmmnv%vnmmmmmmmmmmnv%vnmmmnv%vnmmmnv 
  vnmmnv%vnmmmmmnv%vnmm;mmmmmmnv%vnmmmmmmmm;mmnv%vnmmmnv%vnmmmnv, 
 vnmmnv%vnmmmmmnv%vnmm;' mmmmmnv%vnmmmmmmm;' mmnv%vnmmmnv%vnmmmnv 
 vnmmnv%vnmmmmmnv%vn;;    mmmmnv%vnmmmmmm;;    nv%vnmmmmnv%vnmmmnv 
vnmmnv%vnmmmmmmnv%v;;      mmmnv%vnmmmmm;;      v%vnmmmmmnv%vnmmmnv 
vnmmnv%vnmmmmmmnv%vnmmmmmmmmm;;       mmmmmmmmmnv%vnmmmmmmnv%vnmmmnv 
vnmmnv%vnmmmmmmnv%vnmmmmmmmmmm;;     mmmmmmmmmmnv%vnmmmmmmnv%vnmmmnv 
vnmmnv%vnmmmmm nv%vnmmmmmmmmmmnv;, mmmmmmmmmmmmnv%vn;mmmmmnv%vnmmmnv 
vnmmnv%vnmmmmm  nv%vnmmmmmmmmmnv%;nmmmmmmmmmmmnv%vn; mmmmmnv%vnmmmnv 
`vnmmnv%vnmmmm,  v%vnmmmmmmmmmmnv%vnmmmmmmmmmmnv%v;  mmmmnv%vnnmmnv' 
 vnmmnv%vnmmmm;,   %vnmmmmmmmmmnv%vnmmmmmmmmmnv%;'   mmmnv%vnmmmmnv 
  vnmmnv%vnmmmm;;,   nmmm;'              mmmm;;'    mmmnv%vnmmmmnv' 
  `vnmmnv%vnmmmmm;;,.         mmnv%v;,            mmmmnv%vnmmmmnv' 
   `vnmmnv%vnmmmmmmnv%vnmmmmmmmmnv%vnmmmmmmnv%vnmmmmmnv%vnmmmmnv' 
     `vnmvn%vnmmmmmmnv%vnmmmmmmmnv%vnmmmmmnv%vnmmmmmnv%vnmmmnv' 
         `vn%vnmmmmmmn%:%vnmnmmmmnv%vnmmmnv%:%vnmmnv%vnmnv'