Difference between revisions of "Part:BBa K2244009"
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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 ( | + | This part is a functional composite part/device of a light-repressed expression system that is once activated will transcribe the reporter gene mCherry ([https://parts.igem.org/Part:BBa_K2244008 BBa_K2244008]). This device is named the lightOFF system. |
===Biology=== | ===Biology=== | ||
− | - | + | -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. |
-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. | -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. | ||
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-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. | -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 ( | + | -Constitutive promoter ([https://parts.igem.org/Part:BBa_K2244012 BBa_K2244012]), 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 | -T1 terminator ([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]), it is the most used terminator in E. coli system | ||
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===Usage=== | ===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 | + | 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([https://parts.igem.org/Part:BBa_K2244004 BBa_K2244004]) or TorA-opdA( [https://parts.igem.org/Part:BBa_K2244003 BBa_K2244003]) to produce pesticide degrading hydrolase in a light-regulated manner. |
<html> | <html> | ||
<body> | <body> | ||
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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. | 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. | 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. | 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. | ||
Latest revision as of 09:29, 28 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_K2244008). This device is named the lightOFF system.
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.
-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_K2244012), 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_K2244004) or TorA-opdA( BBa_K2244003) to produce pesticide degrading hydrolase in a light-regulated manner.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1015
Illegal NheI site found at 1038 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 845
Illegal AgeI site found at 1753 - 1000COMPATIBLE WITH RFC[1000]