Part:BBa_K1813031
nicX Expression Cassette with LacI Reversed
LacIR nicX
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 105
Illegal BglII site found at 1746 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1895
Illegal AgeI site found at 2123
Illegal AgeI site found at 2321 - 1000COMPATIBLE WITH RFC[1000]
Background of LacI Reversed Ptac NicX 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 nicX. The enzyme coded for by nicX is a maleate isomerase that converts Maleic Acid to Fumaric Acid.[5].
Design and Acquisition
After synthesizing a codon-optimized nicX, we used standard assembly to created a composite part composed of nicX 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 nicX expression cassette was assembled behind a lacI cassette BBa_K1813019 to give us the ability to control the expression of NicX.
All nicX constructs are contained within standard pSB1C3 vectors.
Experience
SDS PAGE Protein Expression for NicX
NicX 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 here and here respectively.
NicX has over-expression at 30°C at the expected size of 42kDa.
Figure 1: 12% SDS-PAGE gel showing expression of a protein sized about 42kDa at 30°C. It is less strongly expressed at other temperatures, uninduced and starter cultures. No band is seen at 42kDa for our "No Insert Control".
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
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