Difference between revisions of "Team:British Columbia/Composite Part"
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| + | <center><h2>Composite Part</h2></center> | ||
| + | <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1813026">BBa_K1813026</a> | ||
| + | <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> | ||
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| + | <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> | ||
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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 nicCXDFE pathway, which takes 6-HNA to fumarate. Subsequently, fumarate can be used in the microorganism’s central metabolism. </p> | ||
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best New Composite Part award</a>, you must fill out this page. Please give links to the Registry entries for the Composite parts you have made. Please see the Registry's <a href="http://parts.igem.org/Help:Parts#Basic_and_Composite_Parts"> Help:Parts page</a> for more information on part types.</p> | <p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best New Composite Part award</a>, you must fill out this page. Please give links to the Registry entries for the Composite parts you have made. Please see the Registry's <a href="http://parts.igem.org/Help:Parts#Basic_and_Composite_Parts"> Help:Parts page</a> for more information on part types.</p> | ||
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<p>New composite BioBrick devices can be made by combining existing BioBrick Parts (like Inverters, Amplifiers, Smell Generators, Protein Balloon Generators, Senders, Receivers, Actuators, and so on).</p> | <p>New composite BioBrick devices can be made by combining existing BioBrick Parts (like Inverters, Amplifiers, Smell Generators, Protein Balloon Generators, Senders, Receivers, Actuators, and so on).</p> | ||
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Revision as of 02:26, 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.
UBC iGEM 2015