Difference between revisions of "Part:BBa K2043001"
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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>
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This part encodes <b>Catechol-1,2-dioxygenase</b> from <i>Acinetobacter pittii</i>, codon optimized for <i>E. coli</i>. This enzyme is an <b>intradiol dioxygenase</b> that catalyses oxidative ring cleavage of <b>catechol</b>. Anthocyanins, the key pigments of wine, are <b>polyphenolic molecules</b> naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-1,2-dioxygenase a good candidate for <b>anthocyanin degradation</b>. Catechol-1,2-dioxygenase is also found in many species of soil bacteria. In order to facilitate working with this enzyme, we added a C-terminal His-tag for easier purification.
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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>
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<img src="https://static.igem.org/mediawiki/parts/2/2f/Paris_Bettencourt_Catecholase_example.jpg">
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<b>Figure 1</b>
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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>
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Figure 1 shows the action of Catechol-1,2-dioxygenase in degrading the phenolic ring of catechol, resulting in the opening of this molecule. By similarly degrading the phenol rings of anthocyanins, this enzyme should remove reduce anthocyanin pigmentation.
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In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.<br><br>
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<br><br>
  
 
<b>Testing the part</b><br><br>
 
<b>Testing the part</b><br><br>
  
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We tested the activity of CatA using cell extract of cells expressing our protein. <br>
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We tested the expression and 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><br>
 
First, we performed an SDS-PAGE to check whether the protein was being expressed. <br><br>
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https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg
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<img src="https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg">
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<br><br>
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<br>
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<b>Figure 2</b>
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The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity. <br>
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Although we did not observe expression of the enzyme (Figure 2), we tested the cell extract for CatA activity in 50 mM sodium phosphate at pH 7 using 30 mM catechol as a substrate, following the protocol from the literature (Lin, 2015) (Figure 3).<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>
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The control represents cell extract from <i>E. coli</i> not producing the enzyme. In all cases, values measured correspond to the oxidized catechol reaction product, measured at 260 nm.<br>
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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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<img src="https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg" width=800>
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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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<img src="https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg" width=800><br>
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<b>Figure 3</b><br>
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Based on this result, we can conclude that our codon-optimized enzymes degrades catechol to its reaction product. <br><br>
  
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<b>References</b><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>
 
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>
  
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NCBI Reference Sequence: YP_004995593.1
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Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.<br><br>
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NCBI Reference Sequence: YP_004995593.1<br>
  
 
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Revision as of 22:52, 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 encodes Catechol-1,2-dioxygenase from Acinetobacter pittii, codon optimized for E. coli. This enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-1,2-dioxygenase a good candidate for anthocyanin degradation. Catechol-1,2-dioxygenase is also found in many species of soil bacteria. In order to facilitate working with this enzyme, we added a C-terminal His-tag for easier purification.

Figure 1
Figure 1 shows the action of Catechol-1,2-dioxygenase in degrading the phenolic ring of catechol, resulting in the opening of this molecule. By similarly degrading the phenol rings of anthocyanins, this enzyme should remove reduce anthocyanin pigmentation.

Testing the part

We tested the expression and 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.


Figure 2
Although we did not observe expression of the enzyme (Figure 2), we tested the cell extract for CatA activity in 50 mM sodium phosphate at pH 7 using 30 mM catechol as a substrate, following the protocol from the literature (Lin, 2015) (Figure 3).
The control represents cell extract from E. coli not producing the enzyme. In all cases, values measured correspond to the oxidized catechol reaction product, measured at 260 nm.

Figure 3
Based on this result, we can conclude that our codon-optimized enzymes degrades catechol to its reaction product.

References
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.

Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.

NCBI Reference Sequence: YP_004995593.1