Team:Chiba/protocol/phenotype/timedelay



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Purpose
To Confirm that communication using non-endogenous molecules results in slower activation of receivers.

Equipment

 * shaking incubator(37&deg;C,30&deg;C)
 * Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;C)
 * Taitec BioShaker BR-33FM(30&deg;C,200rpm)
 * 46-well plate(deep well)
 * Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
 * Beckman Allegratm X-12R Centrifuga(Beckman Coulter)

Materials

 * AHL(100&mu;M) solution
 * E.coli Culture Containing BBa_T9002
 * E.coli Culture Containing plasmids you will testing
 * ex.)BBa_K084007(plac+rbs+LasI(no LVA))
 * BBa_K084008(plac+rbs+RhlI(no LVA))


 * If the plasmids you will testing are regulated by IPTG,add IPTG to the reaction culteres when you start the measurement.

Culturing overnight (before the testing day):

 * 1) Inoculate all cultures you will testing from gloycerol stocks into 2 mL of LB-ampicillin liquid medium.
 * 2) Also inoculate a culture containing BBa_T9002 into 2 mL of LB-ampicillin liquid medium.
 * 3) Incubate all cultures with shaking at 37&deg;C(O/N).

Following day

 * cultures containing BBa_T9002
 * 1) Inoculate a culture by adding 100 &mu;L of the cultures into 40mL of LB-ampicillin medium.(in a flask).
 * 2) Incubate a culture for 6-8 hours with shaking at 37&deg;C.
 * 3) Wash
 * 4) Aliquote 10mL of the culture into 50 mL four 50 mL falcon tubes.
 * 5) Cultures are centrifuged at 3500 rpm for 6 minutes.
 * 6) Dinspense supernatant.
 * 7) Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
 * 8) aliquote 1mL of the cultures into a 48-deep well plate(deep well).


 * cultures containing plasmids you will testing
 * 1) Inoculate cultures by adding 12.5 &mu;L of the cultures into 5 mL of LB-ampicillin medium.
 * 2) Incubate cultures for 6-8 hours with shaking at 37&deg;C.
 * 3) Wash
 * 4) Cultures are centrifuged at 3500　rpm for 6 minutes.
 * 5) Dinspense supernatant.
 * 6) Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
 * 7) repeat washing process twice.
 * 8) Cultures are centrifuged at 3500　rpm for 6 minutes.
 * 9) Dinspense supernatant.
 * 10) Add 5　mL of new LB-ampicillin medium and resuspense with pipetting.
 * 11) aliquote cultures into a 48-deep well plate(deep well).

Measurement
ex.)sender culture:500&mu;L,receiver culture:500&mu;L
 * Mix senders culture and receiver culture(Prepare three replicate cultures).
 * Positive control:receiver culture with 100&mu;M AHL solution.
 * Negative control:receiver culture without sender culture and AHL solution.

return to Sender experiments details
 * 1) Incubate testing cultures with shaking at 37&deg;C.
 * 2) After some intervals,aliqupte 100&mu;L of testing cultures into a 96-well plate(shallow well).
 * 3) Measure fluorescence intensity.
 * conditions
 * shaking(before measurement):On time = 1min,Off time = 10 sec,
 * integration time = 1000 ms
 * Beam width:Normal Beam
 * Wavelength pair = 485 nm(excitation) and 527 nm(emission)