Part:BBa_K2244007

ColE promoter +mhei gene +mcherry gene +T1terminator

The device is a functional composite part containing a mheI-mCherry fusion protein .

Biology

-Co1E promoter (BBa_K2244006) is derived from the promoter region of colicin E gene located in the ColE1 plasmid of E.coli. ColE promoter contains a ‘SOS’ operator region that allows the binding of LexA protein to repress transcription. DNA-binding component of LexA repressor in LEV1 would form a dimer and bind to the operator sequence thus halts the activity of ColE promoter. -mheI-mCherry is a coding sequence for a fusion protein. mheI (BBa_K2244004) encodes MBC-hydrolyzing esterase (MHE) that degrades MBC (carbendazim) pesticide. MHE is responsible for carrying out the first step detoxification (MBC to 2-AB), without the need of any cofactor. mCherry (BBa_K2244008) is a red fluorescent protein used as a reporter gene. This fusion protein allows the visulization of the part being uptake by cells. - -T1 terminator (BBa_B0010), it is the most used terminator in E. coli system.

Usage

In our project this year, this device worked in the lightOFF system (BBa_k2244009) to replace the ColE promoter+mCherry+T1 terminator section and to allow the expression of MHE when induced in darkness. To demonstrated the functionality of this part, we performed ELISA and enzymatic activity studies (Figure 1-3) to prove that functional MHE was successfully produced using light-regulated system.

 Figure: ELISA studies of OPH protein expression in periplasmic fraction, cytoplasmic fraction, whole cell, and periplasmic fraction of pLEV1(408) (control strain).

Figure: Specific OPH activities of whole cell, periplasmic fraction, cytoplasmic fraction and control periplasmic fraction (lightOFF). The activity was assayed with paraoxon as substrate. Data are mean values+/-standard derivations from three replicates.

Figure illustration of OPH activity vs various concentrations of periplasmic fractions from OPH-expressed cell strain (black) and control strain (Red). The activity was assayed with paraoxon as substrate. Data are mean values+/-standard derivations from three replicates.

Reference

Aharonson, N. and J. Katan. 1993. Delayed and enhanced biodegradation of soil-applied diphenamid, carbendazim, and aldicarb. Arch. Insect Biochem Physiol. 22,451–466.

Mazellier, P., Leroy, E., De Laat, J. & Legube, B. 2003.Degradation of carbendazim by UV/H2O2 investigated by kinetic modelling. Environ. Chem. Lett. 168–72.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1250
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1250
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 754
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1250
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1250
  Illegal AgeI site found at 413
  
• 1000
  COMPATIBLE WITH RFC[1000]