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| | <partinfo>BBa_K1813001 short</partinfo> | | <partinfo>BBa_K1813001 short</partinfo> |
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| − | ptac rbs cch2 term (in construction)
| + | The enzyme coded for by <i>cch2</i> converts 6-chloronicotinic acid (6-CNA) to 6-Hydronicotinic acid (6-HNA). |
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| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
| | <partinfo>BBa_K1813001 SequenceAndFeatures</partinfo> | | <partinfo>BBa_K1813001 SequenceAndFeatures</partinfo> |
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| | <partinfo>BBa_K1813001 parameters</partinfo> | | <partinfo>BBa_K1813001 parameters</partinfo> |
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| − | <h2>Background of <i>cch2</i></h2>
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| − | <h4>Part Description:</h4>
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| − | <p>
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| − | 6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid degradation that is both toxic to bees (1), and a persistent environmental contaminant (2).The conversion from 6-CNA to 6-HNA, a well studied intermediate in nicotine degradation (3), is catalyzed by 6-chloronicotinic acid chlorohydrolase (cch2), a chlorohydrolase from SG-6C ''Bradyrhizobiaceae'' (4). </p>
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| | + | <h4>Part Function</h4> |
| | + | <p>This is a composite part for <a href="https://parts.igem.org/Part:BBa_K1813026">BBa_K1813026</a>. </p> |
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| − | <h4>Design and Acquisition</h4> | + | <!-- --> |
| − | <p> After synthesizing a codon-optimized cch2, we used standard assembly to created a composite part composed of cch2 driven by the Ptac promoter (BBa_K1813037) and flanked by a double terminator (BBa_B0014).
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| − | The tac promoter contains a lac operator sequence that can be bound by LacI, the lac repressor protein, allowing inducible expression by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Our cch2 expression cassette was assembled behind a lacI cassette (BBa_K1813019) to give us the ability to control the expression of Cch2.
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| − | All cch2 constructs are contained within standard pSB1C3 vectors.</p>
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| − | <h4>Experience</h4>
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| − | <p>
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| − | SDS PAGE Protein Expression for Cch2
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| − | Cch2 was expressed in E.Coli DH5-α.
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| − | Transformed E.Coli was grown at 37°C until an OD600 of 0.6 to 0.8. They were then induced with IPTG and grown overnight at 16°C, 20°C, 25°C, 30°C, 37°C to discern which temperature resulted in optimal protein expression.
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| − | The samples were prepared for SDS page gel via the SDS page sample preparation protocol and SDS page gel protocol (reference).
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| − | cch2 has over-expression at 25°C and 30°C at the expected size of 52kDa, however there is a significant amount of insoluble fraction of the gene product.
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| − | Figure 1:
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| − | 12% SDS-PAGE gel showing expression of a protein sized about 52kDa at 25°C and 30°C. It is less strongly expressed at other temperatures and starter culture, with the exception of 16°C sample.
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| − | Cell Lysate Experiment
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| − | 6-CNA Degradation Rate Experiment
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| − | A 6-CNA degradation rate experiment was performed over a four hour time course in order to better characterize the enzyme. Samples were collected every 15 minutes for 2 hours, and thereafter every 30 minutes for the remaining two hours.
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| − | Figure 2: 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.
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| − | </p>
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| − | References:
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| − | 1. Nauen, R., Ebbinghaus-Kintscher, U. and Schmuck, R. (2001) Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae) Pest. Manag. Sci. 57 (7) DOI: 10.1002/ps.331
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| − | 2. Rouchaud J, Gustin F, Wauters A (1996) Imidacloprid insecticide soil metabolism in sugar beet field crops. Bull Environ Contam Toxicol 56: 29–36. doi: 10.1007/s001289900005
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| − | 3.Tang, H., Yao, Y., Wang, L., Yu, H., Ren, Y. et al. (2012) Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Scientific Reports 2, Article number: 377 doi:10.1038/srep00377
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| − | 4.Shettigar M, Pearce S, Pandey R, Khan F, Dorrian SJ, et al. (2012) Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C. PLoS ONE 7(11): e51162. doi: 10.1371/journal.pone.0051162
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