Part:BBa_K1813026
cch2 Expression Cassette with LacI Reversed
LacI Reversed with cch2.
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]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Background of cch2
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].
Design and Acquisition
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.
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.
All cch2 constructs are contained within standard pSB1C3 vectors.
Experience
SDS PAGE Protein Expression for Cch2
Cch2 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).
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.
Figure 1:
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.
Cell Lysate Experiment
6-CNA Degradation Rate Experiment
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.
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.
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
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