Part:BBa_K1813020
nicC Expression Cassette with LacI Reversed
LacI Reversed nicC
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 105
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1658
- 1000COMPATIBLE WITH RFC[1000]
Background of LacI Ptac NicC 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 nicC. The enzyme coded for by nicC is a 6-HNA monooxygenase that converts 6-Hydroxynicitinoic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP).
Design and Acquisition
After synthesizing a codon-optimized nicC, we used standard assembly to created a composite part composed of nicC driven by the Ptac promoter (BBa_K1813037 ) and flanked by a double terminator (BBa_K1813014 ).
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 nicC expression cassette was assembled behind a lacI cassette (BBa_K1813019 ) to give us the ability to control the expression of nicC.
All nicC constructs are contained within standard pSB1C3 vectors.</p>
Experience
SDS PAGE Protein Expression for nicC
nicC 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). </p>
nicC 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.</p>
Figure 1: 12% SDS-PAGE gel run with nicC. No discernible expression is seen at the expected size of nicC, 43kDa.
References:
<p>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</p>
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</p>
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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