Difference between revisions of "Part:BBa K518007"

Revision as of 18:54, 5 October 2011

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 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-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 and cells transformed with cheZ-expressing plasmid (BBa_K518006).

Our results clearly show that cheZ-deficient mutants show smaller diffusion, while they are rescued by our BBa_K518006 plasmid.

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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.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. A) Graph plot. B) Bar chart plot. Data is expressed as mean±S.D.. N=6.> Sequence and Features