View source for Template:USTC//protocol-AHL

===protocol===
AHLHybrid promoter:BBa_K176026, BBa_K176126, BBa_K176128, BBa_K176130

1.	Streak a plate of the strain which contain one of the parts listed in pSB1A3 .

2.	Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic(Ampicillin 0.1mg/ml) with single colony from the plate.

3.	Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm.

4.	Cultures were diluted 1:1000 to tubes of 3ml fresh medium and grown for 4.5hrs.

5.	Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine is diluted and added to different tubes to yield different final concentrations      (1E-5,1E-7,1E-8,1E-9,1E-10M).To ensure the same response time , the AHL should be added with a time interval of 2mins between tubes, so do the measurements procedure.

6.	Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance ((HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell path length 10mm,600nm,1.5 nm slit width)  for the first time 30 minutes after adding AHL. Repeat measurement every 30 mins in the next 4hrs.