Difference between revisions of "Team:British Columbia/Composite Part"
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<center><h2>Composite Part</h2></center> | <center><h2>Composite Part</h2></center> | ||
| − | <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1813026">BBa_K1813026</a> | + | <center><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1813026">BBa_K1813026 - click for more data</a></center> |
<p>Cch2 is a novel chlorohydrolase that was identified in a soil bacterium, Bradyrhizobiaceae strain SG-6C, that is capable of dechlorinating 6-chloronicotinic acid (6-CNA) to 6-hydroxynicotinic acid (6-HNA). 6-CNA has been found to accumulate in soils and plant material following application of imidacloprid. Due to the associated toxicity of 6-CNA to bees, we designed a construct with cch2 in hopes that heterologous expression in a bee gut microbe, could provide the bee with resistance to the toxic effects of 6-CNA.</p> | <p>Cch2 is a novel chlorohydrolase that was identified in a soil bacterium, Bradyrhizobiaceae strain SG-6C, that is capable of dechlorinating 6-chloronicotinic acid (6-CNA) to 6-hydroxynicotinic acid (6-HNA). 6-CNA has been found to accumulate in soils and plant material following application of imidacloprid. Due to the associated toxicity of 6-CNA to bees, we designed a construct with cch2 in hopes that heterologous expression in a bee gut microbe, could provide the bee with resistance to the toxic effects of 6-CNA.</p> | ||
| − | <p>The cch2 construct was designed with a ribosome binding site, pTac promoter, and LacI repressor to allow for inducible expression. We confirmed expression of cch2 by SDS-PAGE and found maximal expression at 25°C. Furthermore, we demonstrated functionality of Cch2 using whole cell lysate to assay conversion of 6-CNA to 6-HNA. Analysis was performed using GC-MS. A time-course assay with E. coli harboring the cch2 construct was used to determine the conversion rate of 6-CNA to 6-HNA. </p> | + | <p>The <i>cch2</i> construct was designed with a ribosome binding site, pTac promoter, and LacI repressor to allow for inducible expression. We confirmed expression of <i>cch2</i> by SDS-PAGE and found maximal expression at 25°C. Furthermore, we demonstrated functionality of Cch2 using whole cell lysate to assay conversion of 6-CNA to 6-HNA. Analysis was performed using GC-MS. A time-course assay with <i>E. coli</i> harboring the <i>cch2</i> construct was used to determine the conversion rate of 6-CNA to 6-HNA. </p> |
| − | <p>Validation of this part was critical to link the modification of imidacloprid by CYPs with the degradation pathway encoded by the nicCXDFE pathway, which takes 6-HNA to fumarate. Subsequently, fumarate can be used in the microorganism’s central metabolism. </p> | + | <p>Validation of this part was critical to link the modification of imidacloprid by CYPs with the degradation pathway encoded by the <i>nicCXDFE</i> pathway, which takes 6-HNA to fumarate. Subsequently, fumarate can be used in the microorganism’s central metabolism. </p> |
| + | <center><img src="https://static.igem.org/mediawiki/2015/9/9f/Cch2_rate_exp.png"></center> | ||
| + | <p><small>Figure 1: Consumption of 6-CNA and appearance of 6-HNA during 4 hour Resting Cell Assay. Metabolites were identified by GC/MS. The control is E.Coli with an empty PSB 1C3 plasmid. Peaks representing 6-CNA and 6-HNA peaks were in agreement with standards run previously. Degradation of 6-CNA began nearly instantly as the time zero samples had presence of the 6-HNA peak, as seen on the graph. At 60 minutes, all of the 6-CNA had been degraded and only 6-HNA peaks appeared on the GC/MS chromatogram.</small></p> | ||
Latest revision as of 03:37, 19 September 2015
Composite Parts
Composite Part
Cch2 is a novel chlorohydrolase that was identified in a soil bacterium, Bradyrhizobiaceae strain SG-6C, that is capable of dechlorinating 6-chloronicotinic acid (6-CNA) to 6-hydroxynicotinic acid (6-HNA). 6-CNA has been found to accumulate in soils and plant material following application of imidacloprid. Due to the associated toxicity of 6-CNA to bees, we designed a construct with cch2 in hopes that heterologous expression in a bee gut microbe, could provide the bee with resistance to the toxic effects of 6-CNA.
The cch2 construct was designed with a ribosome binding site, pTac promoter, and LacI repressor to allow for inducible expression. We confirmed expression of cch2 by SDS-PAGE and found maximal expression at 25°C. Furthermore, we demonstrated functionality of Cch2 using whole cell lysate to assay conversion of 6-CNA to 6-HNA. Analysis was performed using GC-MS. A time-course assay with E. coli harboring the cch2 construct was used to determine the conversion rate of 6-CNA to 6-HNA.
Validation of this part was critical to link the modification of imidacloprid by CYPs with the degradation pathway encoded by the nicCXDFE pathway, which takes 6-HNA to fumarate. Subsequently, fumarate can be used in the microorganism’s central metabolism.
Figure 1: Consumption of 6-CNA and appearance of 6-HNA during 4 hour Resting Cell Assay. Metabolites were identified by GC/MS. The control is E.Coli with an empty PSB 1C3 plasmid. Peaks representing 6-CNA and 6-HNA peaks were in agreement with standards run previously. Degradation of 6-CNA began nearly instantly as the time zero samples had presence of the 6-HNA peak, as seen on the graph. At 60 minutes, all of the 6-CNA had been degraded and only 6-HNA peaks appeared on the GC/MS chromatogram.
UBC iGEM 2015