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| − | <h4>Part Description:</h4><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]. 6-HNA can be further degraded into Fumaric Acid using the following pathway, which includes <i>nicD</i>. The enzyme coded for by nicD is a N-Formylmaleamate Deformylase that converts N-Formylmaleamic (NFM) to Maleamic Acid, also producing formic acid. [5].</p></p>
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| − | <h4> Design and Acquisition</h4><p> | + | <h4>Part Function:</h4> |
| − | After synthesizing a codon-optimized <i>nicD</i>, we used standard assembly to created a composite part composed of <i>nicD</i> driven by the Ptac promoter
| + | <p> This is a composite part for <a href="https://parts.igem.org/Part: BBa_K1813021"> BBa_K1813021</a>. </ p> |
| − | <html><a href="https://parts.igem.org/Part: BBa_K1813037"> BBa_K1813037</a> </html> and flanked by a double terminator <html><a href="https://parts. igem.org/Part:BBa_B0014">BBa_B0014</ a> </html>. | + | |
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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 <i>nicD</i> expression cassette was assembled behind a lacI cassette <html><a href="BBa_K1813019">BBa_K1813019</a></html> to give us the ability to control the expression of NicD.
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| − | All <i>nicD</i> constructs are contained within standard pSB1C3 vectors.
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| − | <h4>Experience</h4><p>
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| − | SDS PAGE Protein Expression for NicD
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| − | NicD 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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| − | NicD has over-expression at 37°C at the expected size of 30kDa, 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 30kDa at 37°C. It is less strongly expressed at other temperatures and starter culture, with the exception of 16°C sample.
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| − | </p>
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| − | <h4>References:</h4>
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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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| − | <br/>
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| − | [5].Jiménez, J., Canales, A., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J., Díaz, E.(2008) Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440 1Proc Natl Acad Sci 05(32): 11329–11334. doi: 10.1073/pnas.0802273105 PMCID: PMC2516282
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