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Team:Heidelberg/Project/Sensing
From 2008.igem.org
Contents |
Project - Sensing
Abstract
Bacteria have the ability to sense gradients of chemoattractants in their environment by specific receptor molecules which are present in the outer bacteria membrane. Our aim was to make use of bacterial chemotaxis in order to generate a "killer" strain which is able to detect and finally kill a "prey" strain by chemotaxis induced movement towards it. For this purpose we cloned the LuxS gene of Vibrio harveyi in an expression plasmid. LuxS encodes the Autoinducer-2 synthase, an enzyme that is essential for the production of the chemoattractant Autoinducer-2 (AI-2). We wanted to transform the "prey" cells with this expression plasmid in order to establish a concentration gradient of AI-2, which can be sensed by the "killer" strain. To make the "killer" strain sensitive towards AI-2, we generated two different chimeric receptors consisting each of the periplasmic ligand-binding domain of LuxQ, the natural receptor for AI-2, and the cytoplasmic part of the chemotaxis receptor (Tar). To allow proper folding and signal transduction we included either the second transmembrane domain of the LuxQ receptor or the Tar receptor in the chimeric receptor sequence. In addition to the chimeric receptor sequence we wanted to co-express from the same plasmid also LuxP, a cofactor which is essential for AI-2 ligand binding of the LuxQ receptor. We were successful in the cloning of all constructs mentioned above. The chimeric receptors were expressed in transformed bacteria as controlled by receptor fusions to the yellow fluorescent protein YFP. To our surprise none of the chimeric receptor constructs showed a polarized localization as expected for chemotaxis receptors. We were also not able to detect chemotactic activity in the transformed bacteria. Future experiments with sequence optimized receptor fusions will have to show if AI-2 is able to induce Tar-based chemotaxis.
