Endy:F2620/Specificity/Protocols

1. Prepare 1 culture for testing using the standard chassis prepartion protocol.
2. Transfer the appropriate number of 200μl aliquots of each of the cultures into a flat-bottom 96 well plate (Cellstar Uclear bottom, Greiner).
3. Add the eight standard concentrations of each AHL to three replicate aliquots of culture.
4. These plates were incubated in a Wallac Victor3 multi-well fluorimeter at 37C and assayed with an automatically repeating protocol of absorbance measurements (600nm filter, 0.1 second absorbance through approximately 0.5cm of fluid), fluorescence readings (488nm excitation filter, 525nm emission filter, 0.5 second, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed). One well was filled with M9 media in all experiments as an absorbance background. Time between measurements was 3 minutes.

Old protocol details

Day 1:

1. Make overnight culture in 5ml of M9+KAN of T9002 & I7101.3k3.

Day 2:

1. Measure OD of overnight. Record length of time that overnight grew for.
2. Diltue 1:1000 in M9+KAN (30mL M9 & 30μl colony). Dilute into 200ml flask.
3. Set up the protocol (specificity) and heat plate reader to 37C.
4. After 3h measure OD of culture.
5. Put 200μl of culture into wells on a clear-bottomed, 96-well plate. Shake the culture first to ensure it is well-mixed.

1. Well map -

Row	AHL 1x3 repeats	AHL 2x3 repeats	AHL 3x3 repeats	Media
A	0	0	0	media
B	1nM	1nM	1nM	media
C	10nM	10nM	10nM	media
D	100nM	100nM	100nM	empty
E	1uM	1uM	1uM	empty
F	10uM	10uM	10uM	empty
G	100uM	100uM	100uM
H	1mM	1mM	1mM

• Immediately put into plate reader and run protocol

1. Data Analysis

• Plot transfer curves for each

Comments:

• There were difficulties with putting 10AHL, 12AHL and 14AHL into solution. Initial solution of 1mM was prepared by dissolving the AHL powder from Sigma in methanol to the final concentration of 1mM. In order to get it into the solution samples vere vortexed and heated (using Jason's heating block) to 50C. Then the subsequent serial dilutions were into water and there was no problem in diluting the solution. At the time of doing dilution they all seemed to be properly dissolved, but some ppt was observed in 1mM samples after refrigeration the next day.
• 4AHL sample had an outlayer that was removed from data processing. (that was the only outlayer that was removed)