Team:Cambridge/Signalling/Experiments/AGR test
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(New page: =Testing AGR Sender and Reciever= *We can test one, then use to test the other, or we can test them in parallel ==Testing Agr Sender== A number of options have been considered to measure...) |
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*15ml supernantant applied to 3-kDa cutoff membrane. | *15ml supernantant applied to 3-kDa cutoff membrane. | ||
*Filtrate should contain AIP (can be stored at -80°C). Test by applying 50μl of each 10 fold dilution series to 450μl reporter. | *Filtrate should contain AIP (can be stored at -80°C). Test by applying 50μl of each 10 fold dilution series to 450μl reporter. | ||
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Latest revision as of 03:15, 30 October 2008
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Testing AGR Sender and Reciever
Testing Agr SenderA number of options have been considered to measure the AIP output of our sender construct.
The last option is determined to be most feasible. A novel lux-based agrP3-reporter fusion S aureus strain (R0J48) was kindly provided by Rasmus Jensen of Nottingham University (here's the link to the paper http://www.ncbi.nlm.nih.gov/pubmed/18582472). In this strain, the agrABCD locus is knocked out using TetM and lux and the luxABCDE operon inserted downstream of the p3 promoter. agrA and C1 are subsequently reintroduced under the p2 promoter in a plasmid pAgrC1A. Growth conditions:
Until the sender construct is made, we plan to test the reporter strain using alternative sources of AIP. A number of options have been considered:
Again, the last option is most suitable for our investigation. Here's the protocol taken from Balaban et al (http://www.jbc.org/cgi/content/abstract/276/4/2658)
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