Difference between revisions of "Part:BBa K518007"
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<partinfo>BBa_K518007 short</partinfo>
 
<partinfo>BBa_K518007 short</partinfo>
  
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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. With a promoter of your interest, this device rescues the mobility of cheZ-/- cells.
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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. With a promoter of your interest, this device rescues the mobility of ''cheZ''-/- cells.
  
 
===Molecular Biology===
 
===Molecular Biology===
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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).  
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The CheZ intracellular signaling cascade is started by detection of a chemoattractant(1). Four cytoplasmic proteins, CheA, CheW, CheY and CheZ, are involved in this pathway(2).  
  
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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.
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When not stimulated, E. coli flagella rotate CCW (counter-clockwise), so they swim straight. Increasing concentrations of attractant or decreasing concentrations of repellant activate chemoreceptors. When a 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 switch rotation from CCW to CW (clockwise) (3). This change results in the bacteria tumbling.
  
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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).  
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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 possessing this protein (2).
  
 
===Reference===
 
===Reference===
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===Usage===
 
===Usage===
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===Usage===
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We performed a swarming assay to characterize the motility of cheZ-deficient strain and cells transformed with a plasmid containing this device. ([[Part:BBa_K518006 |BBa_K518006]]).
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We performed a swarming assay to characterize the motility of cheZ-deficient strain and cells transformed with cheZ-expressing plasmid ([[Part:BBa_K518006 |BBa_K518006]]).
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Our results clearly show that cheZ-deficient mutants show smaller diffusion, while they are rescued by our [[Part:BBa_K518006 |BBa_K518006]] plasmid.
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Our results clearly show that cheZ-deficient mutants show smaller diffusion, while the motility is rescued by our [[Part:BBa_K518006 |BBa_K518006]] plasmid.
  
 
[[Image:cheZ(-)motility.png|300px]]
 
[[Image:cheZ(-)motility.png|300px]]
  
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<div style="float:left">[[Image:cheZmotility1.png|300px]]</div>
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<div style="float:left">[[Image:cheZmotility1.png|300px]];</div>
 
[[Image:cheZmotility2.png|300px]]
 
[[Image:cheZmotility2.png|300px]]
 
<div style="clear:both"></div>
 
<div style="clear:both"></div>
  
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<div style="float:left">[[Image:cheZmotility3.png|300px]]</div>
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<div style="float:left">[[Image:cheZmotility3.png|300px]];</div>
 
[[Image:cheZmotility4.png|300px]]
 
[[Image:cheZmotility4.png|300px]]
 
<div style="clear:both"></div>
 
<div style="clear:both"></div>
  
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<Fig.1: The diffusion of colonies expressing cheZ in an IPTG-dependent manner. The 2nd to 5th photographs are the representatives of colonies on a 0.25% agar plate containing 1, 10, 40, 100µM IPTG, respectively. The 1st picture is a negative control (non-transformed cheZ-deficient cells). >
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<Fig.1: The diffusion of colonies expressing cheZ in an IPTG-dependent manner. The 2nd to 5th photographs are representatives of colonies on a 0.25% agar plate containing 1, 10, 40, 100µM IPTG, respectively. The 1st picture is a negative control (they are not transformed with this device). >
  
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[[Image:cheZmotilitychart.png|500px]]
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<div style="float:left">[[Image:cheZmotilitychart.png|500px]];</div>
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[[Image:cheZmotilitychart2.png|500px]]
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<Fig.2: The comparison of colony diffusion. Obtained images are thresholded and segmentized. The relative colony size is determined as a pixel number of the segmented region. >
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<div style="clear:both"></div>
  
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<Fig.2: The comparison of colony size. The colony size was an indicator of cell diffusion which we used. The images of colonies were thresholded and segmentized. The relative colony size was determined by the pixel number of the segmented region. A) Graph plot. B) Bar chart plot. Data was expressed as mean±S.D.. n=6.>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 14:07, 30 August 2014

cheZ expression cassette (no promoter)

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. With a promoter of your interest, this device rescues the mobility of cheZ-/- cells.

Molecular Biology

The CheZ intracellular signaling cascade is started by detection of a chemoattractant(1). Four cytoplasmic proteins, CheA, CheW, CheY and CheZ, are involved in this pathway(2).

When not stimulated, E. coli flagella rotate CCW (counter-clockwise), so they swim straight. Increasing concentrations of attractant or decreasing concentrations of repellant activate chemoreceptors. When a 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 switch rotation from CCW to CW (clockwise) (3). This change results in 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 possessing 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 and cells transformed with a plasmid containing this device. (BBa_K518006).

Our results clearly show that cheZ-deficient mutants show smaller diffusion, while the motility is rescued by our BBa_K518006 plasmid.

CheZ(-)motility.png

CheZmotility1.png;

CheZmotility2.png

CheZmotility3.png;

CheZmotility4.png

<Fig.1: The diffusion of colonies expressing cheZ in an IPTG-dependent manner. The 2nd to 5th photographs are representatives of colonies on a 0.25% agar plate containing 1, 10, 40, 100µM IPTG, respectively. The 1st picture is a negative control (they are not transformed with this device). >

CheZmotilitychart.png;

CheZmotilitychart2.png

<Fig.2: The comparison of colony size. The colony size was an indicator of cell diffusion which we used. The images of colonies were thresholded and segmentized. The relative colony size was determined by the pixel number of the segmented region. A) Graph plot. B) Bar chart plot. Data was expressed as mean±S.D.. n=6.> Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]