Difference between revisions of "Part:BBa K2043001"
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<partinfo>BBa_K2043001 short</partinfo>
 
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<span class='h3bb'>Sequence and Features</span>
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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>
 
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>
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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. <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. <br>
 
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product. <br><br>
 
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product. <br><br>
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https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg
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<img src="https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg" width=800>
  
 
As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 260nm, which results from the oxidation of Catechol. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional. <br><br>
 
As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 260nm, which results from the oxidation of Catechol. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional. <br><br>
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2043001 SequenceAndFeatures</partinfo>
 
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Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.<br><br>
 
Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.<br><br>
  
 
NCBI Reference Sequence: YP_004995593.1
 
NCBI Reference Sequence: YP_004995593.1
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Revision as of 21:43, 26 October 2016

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

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. 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.

https://static.igem.org/mediawiki/parts/1/1e/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.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.

As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 260nm, which results from the oxidation of Catechol. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional.

Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.

NCBI Reference Sequence: YP_004995593.1