Difference between revisions of "Part:BBa K2244007"

Latest revision as of 09:31, 28 October 2017

ColE promoter +mhei gene +mcherry gene +T1terminator

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

Biology

-ColE 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]

@@ Line 4: / Line 4: @@
-The device is a functional plasmid containing a MBC hydrolase gene (mheI) ([https://parts.igem.org/Part:BBa_K2244004 BBa_K2244004]), which encodes carbendazim hydrolase. In order to study expression of target gene in cytoplasm, mheI was fused to a reported gene mCherry ([https://parts.igem.org/Part:BBa_J06504 BBa_J06504]), which functions as indicator of the uptake of mheI. This part is regulated under the control of ColE promoter, which is from E. coli ColE1 plasmid.
+The device is a functional composite part containing a mheI-mCherry fusion protein .
 ===Biology===
-ColE promoter([https://parts.igem.org/Part:BBa_K2244006 BBa_K2244006]) sequence 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.
-MBC hydrolase gene (mheI)([https://parts.igem.org/Part:BBa_K2244004 BBa_K2244004]) 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.
+-ColE promoter ([https://parts.igem.org/Part:BBa_K2244006 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 ([https://parts.igem.org/Part:BBa_K2244004 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 ([https://parts.igem.org/Part:BBa_K2244008 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 ([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]), it is the most used terminator in E. coli system.
-mCherry([https://parts.igem.org/Part:BBa_K2244008 BBa_K2244008]) is a red fluorescent protein used as a reporter. It is based on a fluorescent protein that was originally isolated from Discosoma sp.mCherry sequence is codon optimized for E. coli expressionm and can be replaced by any gene sequence in light-regulated studies.
-T1 terminator([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]) is the most commonly used terminator.
+===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.