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<p>When no ligand is bound to the chemotaxis receptor, it activates a signal transduction pathway of several proteins, including CheA, CheY, and CheZ (figure 1). First, CheA phosphorylates CheY, which subsequently translocates to the cell membrane where it binds the flagellar motor protein, thereby altering its rotational direction to a “running” state. After CheY is bound to the flagellar motor protein, it is subsequently dephosphorylated by CheZ. Upon ligand binding, the receptor is inactivated, leading to an inactivated pathway and a rotational direction of the flagellar motor protein in the “tumbling” state. Since the binding of CheY and CheZ is linked to ligand binding of the receptor, measurement of the binding of these proteins by using the BRET assay (described below) provides a quick and accurate indication of the concentration of ligand present (requirement I and II).</p> | <p>When no ligand is bound to the chemotaxis receptor, it activates a signal transduction pathway of several proteins, including CheA, CheY, and CheZ (figure 1). First, CheA phosphorylates CheY, which subsequently translocates to the cell membrane where it binds the flagellar motor protein, thereby altering its rotational direction to a “running” state. After CheY is bound to the flagellar motor protein, it is subsequently dephosphorylated by CheZ. Upon ligand binding, the receptor is inactivated, leading to an inactivated pathway and a rotational direction of the flagellar motor protein in the “tumbling” state. Since the binding of CheY and CheZ is linked to ligand binding of the receptor, measurement of the binding of these proteins by using the BRET assay (described below) provides a quick and accurate indication of the concentration of ligand present (requirement I and II).</p> | ||
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+ | <h4>Chemotaxis pathway modifications to address all considerations</h4> | ||
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+ | <p>To create a biosensor meeting all five listed requirements, the E. coli chemotaxis pathway was customized using a three step approach. First, we set up an assay to verify the extent to which one of the most important chemotaxis receptors, the Tar receptor, could be customized. This enables accurate detection and the possibility to detect diverse ligands (requirement III). Next, a method establishing the ability to measure the activity of this pathway was implemented, using a Bioluminescence Resonance Energy Transfer (BRET)-pair. BRET provides a clear and easily measurable detection signal (requirement II and IV). The last step of the approach facilitates accurate detection at different concentrations, by mimicking receptor methylation. This facilitates detection of different ligand concentrations (requirement I).</p> | ||
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Revision as of 21:53, 12 October 2018