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 the AIP output of our sender construct.

• Using Ellman's reagent to detect thiol groups
• Mass Spec
• Using a biological reporter

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:

• S aureus can be grown in LB or agar plates at 37 degress. The paper suggested the following selection: 10μg/mL Cm, 5μg/mL Ery, 5μg/mL Tet. However, TetM is under control of P2 so expression in the absence of AIP may be too low for TetR. Also, it is unclear how CmR is acquired.
  • Some papers seem to prefer CYGP broth (10 g/liter casamino acids/10 g/liter yeast extract/5 g/liter glucose/5.9 g/liter NaCl/60 mM β-glycerophosphate). (Novick R P. Methods Enzymol. 1991;204:587–636. [PubMed])
• Dilute overnight cultures 1/20 in fresh medium and incubate for 2 h.
• Dilute 1/50 into 96 well plate with dilution series of AIP.
• relative light unit per cell density (RLU/OD492) taken every 30 min for 24 h. Peak value used for each dilution series.
• This can be compared to standard curves generated from known concentraions of AIP given in the paper (fig3). The EC50 of the reporter for AIP-1 is 6±1.

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:

• Chemical synthesis (too difficult)
• Purification using a mini-intein fusion (http://aem.asm.org/cgi/content/abstract/73/19/6036)
• Purification from S.aureus supernatant.

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)

• Post exponential culture (around 200ml) centrifuged at 6000g for 10 minutes at 4°C.
• Supernatant collected and filtered through 0.22μm to remove residual cells.
• Filtrate lyophilized and resuspended in water to one-tenth original volume (10 fold concentrated)
• 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.