Difference between revisions of "Part:BBa K2244009"

Revision as of 10:01, 27 October 2017

ColE promoter+mCherry gene+T1 terminator+Constitutive promoter+Lev1 gene+T1terminator

This part is a functional composite part/device of a light-repressed expression system that is once activated will transcribe the reporter gene mCherry (BBa_). This device is named the lightOFF system

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. -mCherry (BBa_K2244008) is a red fluorescent protein used as a reporter gene. It is based on a fluorescent protein originally isolated from Discosoma sp. In this project, mCherry sequence was codon optimized for E. coli system. -LEV1 repressor (BBa_K2244005) is a fusion protein of VVD and LexA. Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation. LexA repressor is a transcriptional repressor of SOS regulon in E.coli. LEV1 is the core component of this device -Constitutive promoter (BBa_), in this device, it is used to constitutively express Lev1 gene. -T1 terminator (BBa_B0010), it is the most used terminator in E. coli system

Usage

In our project this year, this device worked to express mCherry fluoresence protein under the control of light (Figure). By replacing mCherry with pesticide degrading genes (mheI, BBa_) or (TorA-opdA, BBa_) to produce pesticide degrading hydrolase in a light-regulated manner.

Figure: Cell growth and mCherry expression study in lightOFF system. Wildtype DH5a cells transformed with pLEV1(408) vector. The cells were illuminated with blue light or wrapped in aluminium foil to induce mCherry expression. Fluorescence and OD600 were measured over a 20-h period of time. a.u. arbitary units

Reference

1)Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, et al. (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442. 2)Tabor, J. J., Levskaya, A. & Voigt, C. A, 2011. Multi-chromatic Control of Gene Expression in Escherichia coli. J. Mol.Biol. 405:315–324. 3)Chen, X., Liu, R., Ma, Z., Xu, X, Zhang, H., Xu, J. & Yang, 2016. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Research, 26 (7): 854-7.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1015
Illegal NheI site found at 1038
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 845
Illegal AgeI site found at 1753
1000
COMPATIBLE WITH RFC[1000]

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+This part is a functional composite part/device of a light-repressed expression system that is once activated will transcribe the reporter gene mCherry (BBa_). This device is named the lightOFF system
-The device is a functional plasmid containing a light repressor LEV1 ([https://parts.igem.org/Part:BBa_K2244005 BBa_K2244005]), which undergoes conformational change upon light irradiation and dimerized to bind regulatory sequence on ColE promoter ([https://parts.igem.org/Part:BBa_K2244006 BBa_K2244006]), thus inhibits target gene expression. While in darkness, dimerization does not occur thus gene expression proceeds. This device is a light repressing system with high induction efficiency and low leakage. mCherry (([https://parts.igem.org/Part:BBa_K2244008 BBa_K2244008])) is reporter gene used in this system to prove its function as well as help to characterize it. mCherry gene can be replaced by any gene sequence in light-regulated studies
+Biology
+-Co1E 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.
+-mCherry ([https://parts.igem.org/Part:BBa_K2244008 BBa_K2244008]) is a red fluorescent protein used as a reporter gene. It is based on a fluorescent protein originally isolated from Discosoma sp. In this project, mCherry sequence was codon optimized for E. coli system.
+-LEV1 repressor ([https://parts.igem.org/Part:BBa_K2244005 BBa_K2244005]) is a fusion protein of VVD and LexA. Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation. LexA repressor is a transcriptional repressor of SOS regulon in E.coli. LEV1 is the core component of this device
+-Constitutive promoter (BBa_), in this device, it is used to constitutively express Lev1 gene.
+-T1 terminator ([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]), it is the most used terminator in E. coli system
+Usage
-===Biology===
+In our project this year, this device worked to express mCherry fluoresence protein under the control of light (Figure). By replacing mCherry with pesticide degrading genes (mheI, BBa_) or  (TorA-opdA, BBa_) to produce pesticide degrading hydrolase in a light-regulated manner.
-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.
-mCherry 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.
-LEV1 repressor([https://parts.igem.org/Part:BBa_K2244005 BBa_K2244005]) is a fusion protein of VVD and LexA, LexA repressor is a transcriptional repressor of SOS regulon in E.coli. It’s form chromosome of Escherichia coli str. K-12 substr. MG1655 (strain: K-12, substrain: MG1655) LexA autocleavage, stimulated by RecA, of the first 84 aa of LexA removes the DNA binding region and is required to activate the SOS response.  LexA is a protein that belongs to the LexA family .
-Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation, Based on this property, the VVD LOV domain was fused with a smaller version of the Gal4 DNA binding domain and the p65 transactivation domain. A common feature of several blue light photoreceptors is the presence of LOV domains, which are able to bind a molecule of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as chromophore, forming upon light stimulation a cysteinyl flavin C4a adduct.
-T1 terminator([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]) is the most commonly used terminator in E. coli.
+ Figure: Cell growth and mCherry expression study in lightOFF system. Wildtype DH5a cells transformed with pLEV1(408) vector. The cells were illuminated with blue light or wrapped in aluminium foil to induce mCherry expression. Fluorescence and OD600 were measured over a 20-h period of time. a.u. arbitary units
+Reference
+)Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, et al. (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442.
+)Tabor, J. J., Levskaya, A. & Voigt, C. A, 2011. Multi-chromatic Control of Gene Expression in Escherichia coli. J. Mol.Biol. 405:315–324.
+)Chen, X., Liu, R., Ma, Z., Xu, X, Zhang, H., Xu, J. & Yang, 2016. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Research, 26 (7): 854-7.