Team:Chiba/protocol/phenotype/timedelay

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== Time-delay check==

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

=== Equipments　and Materials ===
====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 Allegra<sup>tm</sup> 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.)[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)])
::[http://partsregistry.org/Part:BBa_K084008 BBa_K084008]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0170 RhlI(no LVA)])
*If the plasmids you will testing are regulated by IPTG,add IPTG when you start the measurement.

=== Protocol ===

====Culturing overnight (before the testing day):====

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

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

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

====Measurement====
*Mix senders culture and receiver culture(Prepare three replicate cultures).
ex.)sender culture:500&mu;L,receiver culture:500&mu;L
::Positive control:receiver culture with 100&mu;M AHL solution.
::Negative control:receiver culture without sender culture and AHL solution.
#Incubate testing cultures with shaking at 37&deg;C.
#After some intervals,aliqupte 100&mu;L of testing cultures into a 96-well plate(shallow well).
#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)
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