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Team:Heidelberg/Notebook/Sensing Group/Cloning
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==Sensing - Cloning strategy== | ==Sensing - Cloning strategy== | ||
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| + | The core part of the sensing project is the construction of the LuxQ-Tar chimeric receptor, which enables the ''E. coli'' killer bacteria to chemotactically respond to a AI-2 gradient and detect prey cells. The quorum-sensing system is amplified from the ''V. harveyi'' genome while the Tar receptor is from ''E. coli''. On the one hand LuxS needs to be cloned and transformed into one cell type, to make them produce and secrete AI-2. On the other hand the periplasmic ligand binding domain is fused to the cytoplasmic domain of Tar and cloned on one plasmid together with LuxP which is necessary for AI-2 binding. Generally, needed restriction sites for cloning are introduced via the PCR primer. | ||
| + | ''In silico'' cloning was performed in SerialCloner [http://serialbasics.free.fr/Serial_Cloner.html SerialCloner], Vector maps were designed with PlasMapper [1] | ||
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| + | === LuxS === | ||
| + | LuxS is amplified from the ''V. harveyi'' genome with primers LuxQa/LuxQc. Subsequently the product is cloned into the pTr99alpha plasmid at the NcoI and BamHI sites and transformed into DH5a competent cells. | ||
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| + | === LuxP === | ||
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| + | === Fusion receptor === | ||
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| + | [1] Dong, X.; Stothard, P.; Forsythe, I. J. & Wishart, D. S., '''PlasMapper: a web server for drawing and auto-annotating plasmid maps''', Nucleic Acids Res, Vol. 32, pp. W660-W664, 2004 | ||
Revision as of 21:00, 26 October 2008
Contents |
Sensing - Cloning strategy
The core part of the sensing project is the construction of the LuxQ-Tar chimeric receptor, which enables the E. coli killer bacteria to chemotactically respond to a AI-2 gradient and detect prey cells. The quorum-sensing system is amplified from the V. harveyi genome while the Tar receptor is from E. coli. On the one hand LuxS needs to be cloned and transformed into one cell type, to make them produce and secrete AI-2. On the other hand the periplasmic ligand binding domain is fused to the cytoplasmic domain of Tar and cloned on one plasmid together with LuxP which is necessary for AI-2 binding. Generally, needed restriction sites for cloning are introduced via the PCR primer. In silico cloning was performed in SerialCloner [http://serialbasics.free.fr/Serial_Cloner.html SerialCloner], Vector maps were designed with PlasMapper [1]
LuxS
LuxS is amplified from the V. harveyi genome with primers LuxQa/LuxQc. Subsequently the product is cloned into the pTr99alpha plasmid at the NcoI and BamHI sites and transformed into DH5a competent cells.
LuxP
Fusion receptor
[1] Dong, X.; Stothard, P.; Forsythe, I. J. & Wishart, D. S., PlasMapper: a web server for drawing and auto-annotating plasmid maps, Nucleic Acids Res, Vol. 32, pp. W660-W664, 2004
