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===Usage===
 
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A light-repressed gene expression system was used to express MHE in the darkness induction.
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mheI sequence is 729bp in length. Fig 1 shows PCR amplification product of mheI coding region. In the current project, mheI gene was expressed in a light-repressed system (LightOFF), and was induced in darkness to produce MHE. Enzymatic studies of MHE using carbendazim as a substrate. Fig 2 showed that MHE expressed from lightOFF system in darkness-induced cells was able to hydrolyze carbendazim, while the controls, including the non-induced, heat-inactivated MHE, and lightOFF vector, did not possess carbendazim degradation ability. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1μmol of MBC to 2-AB at 30℃ within 1 min.
 
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<center><b>Figure 1:</b> The agarose gel electrophoresis of mheI PCR product
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<center><img src="https://static.igem.org/mediawiki/parts/7/73/MheI.png" style=" width:20%" /> </center>;
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<center><b>Figure 1:</b> Specific MHE enzyme activities of total protein fraction (mheI dark) and control protein fractions (inactive mheI, mheI light, LightOFF). The activity was assayed with carbendazim (MBC) as substrate. Data are mean values+/-standard derivations from three replicates.
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Revision as of 02:22, 31 October 2017

mheI


Biology

This part is a functional gene encoding a Carbendazim (Methyl-1H-Benzimidazol-2-ylcarbamate, or MBC) pesticide hydrolyzing esterase. mheI encodes MBC-hydrolyzing esterase (MHE) that degrades MBC pesticide. Degradation of MBC is achieved by hydrolyzing MBC to 2-aminobenzimidazole (2-AB), which is then converted to benzimidazole or 2-hydroxybenzimidazole (2-HB). The conversion of 2-AB was inhibited by NH4NO3. The benzene ring of 2-HB was further opened through meta catechol cleavage. MHE is responsible for carrying out the first step detoxification (MBC to 2-AB), without the need of any cofactor. MHE is a soluble serine hydrolase of 242 amino acid residues and has a molecular size of 25-27 kDa. MBC hydrolase gene (mheI) is derived from carbendazim-degrading strain Mycobacterium sp. SD-4 and is a genomic DNA gene sequence. mheI gene is also evolutionary conserved in many other bacteria strains, such as Nocardioides sp. SG-4G, and Rhodococcus erythropolis.


Usage

mheI sequence is 729bp in length. Fig 1 shows PCR amplification product of mheI coding region. In the current project, mheI gene was expressed in a light-repressed system (LightOFF), and was induced in darkness to produce MHE. Enzymatic studies of MHE using carbendazim as a substrate. Fig 2 showed that MHE expressed from lightOFF system in darkness-induced cells was able to hydrolyze carbendazim, while the controls, including the non-induced, heat-inactivated MHE, and lightOFF vector, did not possess carbendazim degradation ability. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1μmol of MBC to 2-AB at 30℃ within 1 min.

;

Figure 1: The agarose gel electrophoresis of mheI PCR product

;

Figure 1: Specific MHE enzyme activities of total protein fraction (mheI dark) and control protein fractions (inactive mheI, mheI light, LightOFF). The activity was assayed with carbendazim (MBC) as substrate. Data are mean values+/-standard derivations from three replicates.


Reference

Xu, J.L., Gu, X.Y., Shen, B., Wang, Z.C., Wang, K. & Li, S.P. 2006. Isolation and characterization of a carbendazim-degrading Rhodococcus sp. djl-6. Curr. Microbiol. 53 (1), 72–76.

Zhang, X.J., Huang, Y. J., Harvey, P.R., Li, H.M. Ren, Y., Li, J.S., Wang, J.N. & Yang, H.T. 2013. Isolation and characterization of carbendazim-degrading rhodococcus erythropolis djl-11. PLoS One 8.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 591
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 250
  • 1000
    COMPATIBLE WITH RFC[1000]