Team:Calgary/Lab

From 2009.igem.org

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THE LAB PROJECT
THE LAB PROJECT
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The wetlab aspect for our project aims to engineer and characterize a novel Autoinducer-2 (AI-2) signalling system in E. coli for the study of quorum sensing in Gram-negative bacteria. Quorum sensing is a process where microorganisms use molecules to monitor their own population density and communicate with different species. Different applications have been developed through quorum sensing, including induction of virulence, biofilm formation and genetic competence. Our team decided to choose this signalling system because it has been shown that over 50% of bacteria whose genomes are sequenced to date are able to make this specific signal for pathogenic activity.
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<div class="heading">General Model</div>
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In Vibrio harveyi, the quorum sensing signal is induced by a molecule called AI-2. AI-2 is bound to the protein LuxP in the periplasm, and the LuxP-AI-2 complex interacts with another membrane-bound sensor histidine kinase, LuxQ. At low cell density, with low amounts of autoinducers, LuxQ sensor acts as a kinase, autophosphorylates and transfers the phosphate to the cytoplasmic protein LuxU. LuxU transfers the phosphate to the DNA binding response regulator protein LuxO. Phosphorylated LuxO with a transcription factor _54, activates the transcription of genes encoding five regulatory small RNAs (sRNAs) termed Qrr1-5. The sRNAs bind to and destabilize the mRNA encoding the transcriptional activator, LuxR4, which is needed to activate the transcription of the luciferase operon, luxCDABE. Therefore, when there is a low cell density, the Lux R mRNA is degraded, thus the bacteria do not express bioluminescence. At higher cell density, when AI-2 concentration reaches the threshold, LuxQ sensor switches from a kinase to a phosphatase and removes the phosphate from Lux O via Lux U. Unphosphorylated LuxO cannot induce the expression of the sRNA, which in turn allows translation of LuxR mRNA, the production of Lux R and the expression of bioluminescence.
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Revision as of 22:49, 23 July 2009

University of Calgary

UNIVERSITY OF CALGARY



LAB INDEX
Overview
Updates
Notebook
-LuxPQ
-LuxOU
-LuxCDABE
-pqrr4
-LuxOD47A
-LuxOD47E
-Dispersin
Protocol & Safety
THE LAB PROJECT
The wetlab aspect for our project aims to engineer and characterize a novel Autoinducer-2 (AI-2) signalling system in E. coli for the study of quorum sensing in Gram-negative bacteria. Quorum sensing is a process where microorganisms use molecules to monitor their own population density and communicate with different species. Different applications have been developed through quorum sensing, including induction of virulence, biofilm formation and genetic competence. Our team decided to choose this signalling system because it has been shown that over 50% of bacteria whose genomes are sequenced to date are able to make this specific signal for pathogenic activity.

General Model
In Vibrio harveyi, the quorum sensing signal is induced by a molecule called AI-2. AI-2 is bound to the protein LuxP in the periplasm, and the LuxP-AI-2 complex interacts with another membrane-bound sensor histidine kinase, LuxQ. At low cell density, with low amounts of autoinducers, LuxQ sensor acts as a kinase, autophosphorylates and transfers the phosphate to the cytoplasmic protein LuxU. LuxU transfers the phosphate to the DNA binding response regulator protein LuxO. Phosphorylated LuxO with a transcription factor _54, activates the transcription of genes encoding five regulatory small RNAs (sRNAs) termed Qrr1-5. The sRNAs bind to and destabilize the mRNA encoding the transcriptional activator, LuxR4, which is needed to activate the transcription of the luciferase operon, luxCDABE. Therefore, when there is a low cell density, the Lux R mRNA is degraded, thus the bacteria do not express bioluminescence. At higher cell density, when AI-2 concentration reaches the threshold, LuxQ sensor switches from a kinase to a phosphatase and removes the phosphate from Lux O via Lux U. Unphosphorylated LuxO cannot induce the expression of the sRNA, which in turn allows translation of LuxR mRNA, the production of Lux R and the expression of bioluminescence.

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