Difference between revisions of "Part:BBa K518008"
| (3 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| − | |||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K518008 short</partinfo> | <partinfo>BBa_K518008 short</partinfo> | ||
| Line 6: | Line 5: | ||
This device usually allows strong cheZ expression, while in the presense of IPTG, the transcription of cheZ is repressed by the overexpression of lambda cI repressor BBa_C0051. | This device usually allows strong cheZ expression, while in the presense of IPTG, the transcription of cheZ is repressed by the overexpression of lambda cI repressor BBa_C0051. | ||
| − | + | ===Molecular Biology=== | |
| − | === | + | Once the receptors detect either a chemoattractant or a chemorepellant, they undergo a conformational change and start an intracellular signaling cascade(1). Four cytoplasmic proteins, CheA, CheW, CheY and CheZ, are involved in this pathway(2). |
| + | |||
| + | When not stimulated, E. coli flagella rotate CCW (counter-clock wise), so they swim straight. Increasing concentrations of attractant and decreasing concentrations of repellant causes the chemoreceptors to be active. When the repellant binds, the CheA (a histidine kinase) and CheW proteins bind to the cytoplasmic domain of the receptor protein, the binding causing CheA to be phosphorylated (1). Phosphorylated CheA phosphorylates CheY protein which then binds to the motor of the flagellum, making it to switch rotation from CCW to CW (clock wise) (3). This change results the bacteria tumbling. | ||
| + | |||
| + | Work done by Welch et al. (3) showed that CheY protein is active only when phosphorylated. The dephosphorylation and inactivation of CheY is regulated by CheZ protein (1). It has been suggested that mutants lacking CheZ tumble more often than those having this protein (2). | ||
| + | |||
| + | ===Reference=== | ||
| + | 1. Alberts et al. (1992) Cell Signaling Cell Biology Chapter 15:773-778. | ||
| + | |||
| + | 2. Parkinson J. S.(1993) Signal transduction Schemes of Bacteria [Review]. Cell . 73(5): 857-871. | ||
| + | |||
| + | 3. Welch, M. et al.(1993) Phosphorylation Dependent Binding of a Signal Molecule to the Flagellar Switch of Bacteria. Proc. Natl. Acad. Sci. 90: 8787-8791. | ||
| + | |||
| + | ===Usage=== | ||
| + | We performed a swarming assay to characterize the motility of cheZ-deficient strain transformed with either [[Part:BBa_R0051 |BBa_R0051]] + [[Part: BBa_K518007 |BBa_K518007]], or [[Part:BBa_K518008 |BBa_K518008]]. | ||
| + | |||
| + | Our results clearly show that rescued-cheZ mutants present extensive diffusion, while cheZ repression deprives transformed cells of their motility. | ||
| + | |||
| + | <div style="float:left">[[Image:c1p_cheZmotility1.png|300px]];</div> | ||
| + | [[Image:c2p_cheZmotility2.png|300px]] | ||
| + | <div style="clear:both"></div> | ||
| + | |||
| + | <Fig.1: The diffusion of colonies expressing cheZ was stopped . The 1st picture is a positive control (cheZ-deficient cells transformed with R0051-K518007). The 2nd photograph is the representative of [[Part:BBa_K518008 |BBa_K518008]] colonies on a 0.25% agar plate containing 100µM IPTG. Cell diffusion was clearly repressed. > | ||
| + | |||
| + | <div style="float:left">[[Image:c1p_cheZmotilitychart1.png|500px]];</div> | ||
| + | [[Image:c1p_cheZmotilitychart2.png|500px]] | ||
| + | <div style="clear:both"></div> | ||
| + | |||
| + | <Fig.2: The comparison of colony diffusion. Obtained images are thresholded and segmentized. The arbitrary colony size is determined as a pixel number of the segmented region. Data is expressed as mean±S.D.. N=6.> | ||
| + | |||
| + | For experimental details, see [http://2011.igem.org/Team:UT-Tokyo/Project/Results Result Page]. | ||
<!-- --> | <!-- --> | ||
Latest revision as of 18:24, 5 October 2011
IPTG-inducible CheZ repression device
CheZ is responsible for the dephosphorylation of flagellum-regulating protein CheY. It has been reported that cheZ-/- strain has a higher frequency of direction change and thus a narrower range of mobility. This device usually allows strong cheZ expression, while in the presense of IPTG, the transcription of cheZ is repressed by the overexpression of lambda cI repressor BBa_C0051.
Molecular Biology
Once the receptors detect either a chemoattractant or a chemorepellant, they undergo a conformational change and start an intracellular signaling cascade(1). Four cytoplasmic proteins, CheA, CheW, CheY and CheZ, are involved in this pathway(2).
When not stimulated, E. coli flagella rotate CCW (counter-clock wise), so they swim straight. Increasing concentrations of attractant and decreasing concentrations of repellant causes the chemoreceptors to be active. When the repellant binds, the CheA (a histidine kinase) and CheW proteins bind to the cytoplasmic domain of the receptor protein, the binding causing CheA to be phosphorylated (1). Phosphorylated CheA phosphorylates CheY protein which then binds to the motor of the flagellum, making it to switch rotation from CCW to CW (clock wise) (3). This change results the bacteria tumbling.
Work done by Welch et al. (3) showed that CheY protein is active only when phosphorylated. The dephosphorylation and inactivation of CheY is regulated by CheZ protein (1). It has been suggested that mutants lacking CheZ tumble more often than those having this protein (2).
Reference
1. Alberts et al. (1992) Cell Signaling Cell Biology Chapter 15:773-778.
2. Parkinson J. S.(1993) Signal transduction Schemes of Bacteria [Review]. Cell . 73(5): 857-871.
3. Welch, M. et al.(1993) Phosphorylation Dependent Binding of a Signal Molecule to the Flagellar Switch of Bacteria. Proc. Natl. Acad. Sci. 90: 8787-8791.
Usage
We performed a swarming assay to characterize the motility of cheZ-deficient strain transformed with either BBa_R0051 + BBa_K518007, or BBa_K518008.
Our results clearly show that rescued-cheZ mutants present extensive diffusion, while cheZ repression deprives transformed cells of their motility.
;
<Fig.1: The diffusion of colonies expressing cheZ was stopped . The 1st picture is a positive control (cheZ-deficient cells transformed with R0051-K518007). The 2nd photograph is the representative of BBa_K518008 colonies on a 0.25% agar plate containing 100µM IPTG. Cell diffusion was clearly repressed. >
<Fig.2: The comparison of colony diffusion. Obtained images are thresholded and segmentized. The arbitrary colony size is determined as a pixel number of the segmented region. Data is expressed as mean±S.D.. N=6.>
For experimental details, see [http://2011.igem.org/Team:UT-Tokyo/Project/Results Result Page].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]