Difference between revisions of "Part:BBa K2043001"
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− | This part | + | 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. |
− | In order to facilitate working with this enzyme, we added a | + | <br> |
+ | <img src="https://static.igem.org/mediawiki/parts/2/2f/Paris_Bettencourt_Catecholase_example.jpg"> | ||
+ | <br> | ||
+ | <b>Figure 1</b> | ||
+ | <br> | ||
− | + | 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. | |
− | + | <br><br> | |
<b>Testing the part</b><br><br> | <b>Testing the part</b><br><br> | ||
− | We tested the activity of CatA using cell extract of cells expressing our protein. <br> | + | 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> | ||
− | https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg | + | <img src="https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg"> |
− | <br><br> | + | <br> |
+ | <b>Figure 2</b> | ||
+ | <br> | ||
− | + | 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> | |
− | + | 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> | |
− | + | ||
− | + | ||
− | + | <img src="https://static.igem.org/mediawiki/parts/6/63/Paris_Bettencourt_notebook_catA_good.jpg" width=800><br> | |
+ | <b>Figure 3</b><br> | ||
+ | Based on this result, we can conclude that our codon-optimized enzymes degrades catechol to its reaction product. <br><br> | ||
+ | <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> | ||
− | NCBI Reference Sequence: YP_004995593.1 | + | Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.<br><br> |
+ | |||
+ | 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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