Difference between revisions of "Team:British Columbia/Composite Part"

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<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>
 
<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>
  
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<img src="https://static.igem.org/mediawiki/2015/9/9f/Cch2_rate_exp.png">
  
  

Revision as of 03:32, 19 September 2015

UBC iGEM 2015

 

Composite Parts

 

Composite Part

BBa_K1813026 - click for more data

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.