Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/03/21

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Picked 2 E0240 colonies into 2ml. Checked the plates that I pipetted S-R pairs onto last night under UV, didn't see anything. Pipetted the co-cultures onto the plate (15-20ul). Didn't see anything. Miniprepped at 6ish. low yields. Cut E0240 with X/P, ran on gel.

Grew from 11am-6 or 7pm. Spun down co-cultures at 12.5ish for 2mins. Pours off supernatant, resuspended in ~100ul H2) (ended up being ~150 total with residual LB and cells). Used plate reader to check fluorescence for CFP and got higher readings for RhlI-RhlR.

RhlR onlyLuxI-RhlRLasI-RhlRRhlI-RhlR
40515398184032547768


ligation of K092900-E0240

K0929004ul of 12ng/ul
E0240 14ul (didn't check conc, was faint on gel)
2x buffer20ul
T4 ligase 1ul

Transformed into 50ul of BL21. At RT for 10min, ice for 13min, 42deg for 45s
New experiment: grow separate, add sups, let grow. measure at different time points. 1ml for each measurement. Will need to spin will to remove all sender cells. Run in triplicate.

LuxILasIRhlIRhlR only
1hr
3hr
5hr
7hr

4-6ml cultures of senders 3-35ml cultures of RhlR Started at 11:30PM


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