Difference between revisions of "Part:BBa K2317003"

 
 
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<partinfo>BBa_K2317003 short</partinfo>
 
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Catechol 1,2-dioxygenase,responsible for ring cleavage in aromatic compounds degrading.
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cphA-1 is a catechol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6, and isresponsible for ring cleavage in aromatic compounds degrading.
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===Usage and Biology===
 
===Usage and Biology===
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<partinfo>BBa_K2317003 parameters</partinfo>
 
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<h3>Description</h3>
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<p>It was found that the cphA-1 enzyme could catalyze the degradation of catechol, 4-chlorocatechol, and 3-methylcatechol. </p><br>
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<p>To certify success in constructing pSB1C3-cphA-1, 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 915bp of cphA-1.</p><br/>
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<img src="https://static.igem.org/mediawiki/parts/e/ef/T--Jilin_China--cphA-1-01.png" width="250" /><br/>
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Figure 1. Digestion assay of pSB1C3-cphA-1.
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<p>In our project, cphA-1 gene is cloned into expression vector pET28a and induced by IPTG. The optimized induction condition is 0.2mM IPTG, 180rpm in 16℃ for 24h. The enzyme is further purified by affinity chromatography.</p><br/>
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<p>CphA-1 activity was determined by measuring the increase of absorbance at 260 nm in 37℃, corresponding to the maximum absorbance of product muconic acid.</p>
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Latest revision as of 00:49, 2 November 2017

cphA-1

cphA-1 is a catechol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6, and isresponsible for ring cleavage in aromatic compounds degrading.
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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 214
    Illegal NgoMIV site found at 391
    Illegal NgoMIV site found at 757
  • 1000
    COMPATIBLE WITH RFC[1000]


Description

It was found that the cphA-1 enzyme could catalyze the degradation of catechol, 4-chlorocatechol, and 3-methylcatechol.


To certify success in constructing pSB1C3-cphA-1, digestion assay using Eco RI and Pst I was performed. The product digested by Eco RI and Pst I was around 900bp as predicted 915bp of cphA-1.



Figure 1. Digestion assay of pSB1C3-cphA-1.

In our project, cphA-1 gene is cloned into expression vector pET28a and induced by IPTG. The optimized induction condition is 0.2mM IPTG, 180rpm in 16℃ for 24h. The enzyme is further purified by affinity chromatography.


CphA-1 activity was determined by measuring the increase of absorbance at 260 nm in 37℃, corresponding to the maximum absorbance of product muconic acid.