Difference between revisions of "Part:BBa K1813021"

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nicD Expression Cassette with LacI Reversed

Sequence and Features

Background of LacI Ptac NicD Term

Part Description

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 nicD. The enzyme coded for by nicD is a N-Formylmaleamate Deformylase that converts N-Formylmaleamic (NFM) to Maleamic Acid, also producing formic acid [5].

Design and Acquisition

After synthesizing a codon-optimized nicD, we used standard assembly to created a composite part composed of nicD driven by the Ptac promoter BBa_K1813037 and flanked by a double terminator BBa_B0014.

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 nicD expression cassette was assembled behind a lacI cassette BBa_K1813019 to give us the ability to control the expression of NicD.

All nicD constructs are contained within standard pSB1C3 vectors.

Experience

SDS PAGE Protein Expression for NicD

NicD was expressed in E.Coli DH5-α.

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.

The samples were prepared for SDS page gel via the SDS page sample preparation protocol and SDS page gel protocol (reference).

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.

Figure 1: 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.

References

[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
[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
[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
[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
[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