Difference between revisions of "Part:BBa K2317004"

 
 
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Hydroxyquinol 1,2-dioxygenase,responsible for ring cleavage in aromatic compounds degrading.
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CaO19 is a eukaryotic catechol 1,2-dioxygenase (1,2-CTD)and was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity.<br>
  
 
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<h3>Description</h3>
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<p>To certify success in constructing pSB1C3-CaO19, digestion assay using <i>Eco</i> RI and Pst I was performed. The product digested by <i>Eco</i> RI and Pst I was around 900bp as predicted 912bp of CaO19.</p><br/>
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<img src="https://static.igem.org/mediawiki/parts/c/cc/T--Jilin_China--CaO19-01.png" width="300" /><br/>
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Figure 1. Digestion assay of pSB1C3-CaO19.
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<p>In our project, Cao19 gene is cloned into expression vector pET28a and induced by IPTG. The optimized induction condition is 0.3mM IPTG, 180rpm in 25℃ for 16h. The enzyme is further purified by affinity chromatography.</p><br/>
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<p>Cao19 activity was determined by measuring the increase of absorbance at 260 nm, corresponding to the maximum absorbance of product muconic acid. The standard assay of enzyme activity was performed by addition of 2μ (68g/ml) of 1,2-CTD to 998μ of assay solution (PBS containing 20M FeSO4 and 0.1 mM of catechol) at 25°C.</p>
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Latest revision as of 01:09, 2 November 2017

CaO19

CaO19 is a eukaryotic catechol 1,2-dioxygenase (1,2-CTD)and was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 558


Description

To certify success in constructing pSB1C3-CaO19, digestion assay using Eco RI and Pst I was performed. The product digested by Eco RI and Pst I was around 900bp as predicted 912bp of CaO19.



Figure 1. Digestion assay of pSB1C3-CaO19.

In our project, Cao19 gene is cloned into expression vector pET28a and induced by IPTG. The optimized induction condition is 0.3mM IPTG, 180rpm in 25℃ for 16h. The enzyme is further purified by affinity chromatography.


Cao19 activity was determined by measuring the increase of absorbance at 260 nm, corresponding to the maximum absorbance of product muconic acid. The standard assay of enzyme activity was performed by addition of 2μ (68g/ml) of 1,2-CTD to 998μ of assay solution (PBS containing 20M FeSO4 and 0.1 mM of catechol) at 25°C.