Difference between revisions of "Part:BBa K2317003"
| Line 23: | Line 23: | ||
<h3>Description</h3> | <h3>Description</h3> | ||
<p>It was found that the cphA-1 enzyme could catalyze the degradation of catechol, 4-chlorocatechol, and 3-methylcatechol. </p><br> | <p>It was found that the cphA-1 enzyme could catalyze the degradation of catechol, 4-chlorocatechol, and 3-methylcatechol. </p><br> | ||
| − | <p>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.</p><br/> | + | <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 Eco RI and Pst I was around 900bp as predicted 915bp of cphA-1.</p><br/> |
<div class="pic_box center"> | <div class="pic_box center"> | ||
<img src="https://static.igem.org/mediawiki/parts/e/ef/T--Jilin_China--cphA-1-01.png" width="250" /><br/> | <img src="https://static.igem.org/mediawiki/parts/e/ef/T--Jilin_China--cphA-1-01.png" width="250" /><br/> | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 214
Illegal NgoMIV site found at 391
Illegal NgoMIV site found at 757 - 1000COMPATIBLE 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.