Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/05/30

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Put 1ml of each supernatant transfer experiment and co-culture experiment into a 2ml tube and put at 4deg. Left the rest shaking overnight (~2ml left for each sup exp and ~1ml left for each co-culture exp).
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9AM

BL21 sender cultures grew in 6mls and 2mls. RhlR-GFP still didn't grow.
Shook LuxR, TraI, and TraR that had been in the fridge for a few minutes.

Co-cultures: Add 100ul of Sender, 100ul of Receiver to 3ml LB+Amp (except for TraI culture, which will not have antibiotic). Started shaking at 10AM.

SenderReceiver
LuxILuxR
LasILuxR
RhlILuxR
TraILuxR
TraR ctrlLuxR



Supernatant transfer: Spun down 4ml of culture in 2-2ml tubes for each sender. Poured supernatant into round bottom tubes. Dropped some supernatant onto pH strip. Added 1ml of receiver cells to a 2ml centrifuge tube. Added 20ul of Amp and shook. Added 100ul of Receivers + (~4ul) Amp to each 4ml volume of Sender cells in round bottom tubes. Added 1ml of LB+Amp to add nutrients and lower the pH a little. Same Sender-Receiver pairs as above. Started shaking ~10:30AM.
Determine pH using test strips:

SenderpH
Plain LB6-7
LuxI7-8
LasI7-8
RhlI7-8
TraI7-8
TraR ctrl7-8



7:30PM Spun 1ml of supernatant transfer experiments and co-culture experiments in 2ml tubes at full speed for 3 minutes. Poured off supernatant, resuspended cells in 100ul water. Could see that some of the cell pellets were green while controls were not. Moved resuspended cells to plate for fluorescence reading. Added 100ul of each supernatant culture to plate as well for cell density readings.

Supernatant transfer results for LuxR receiver:




Put 1ml of each supernatant transfer experiment and co-culture experiment into a 2ml tube and put at 4deg. Left the rest shaking overnight (~2ml left for each sup exp and ~1ml left for each co-culture exp).


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