Template:USTC//protocol-AHL+aTc

protocols

aTc,AHLHybrid promoter: BBa_K176070, BBa_K176071, BBa_K176075, BBa_K176078:

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 11 tubes 3ml fresh medium and grown for 4.5hrs.

5. Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine and aTc (anhydrotetracycline) are diluted and added to different tube to get different final concentration listed in the table below:

Tube No.	1	2	3	4	5	6	7	8	9	10	11
c(AHL)/M	0	1.00E-06	1.00E-06	1.00E-06	1.00E-06	1.00E-06	1.00E-04	1.00E-04	1.00E-04	1.00E-04	1.00E-04
C(Atc)/ng/ml	0	0	2	20	200	2000	0	2	20	200	2000

To ensure the same response time , the AHL and aTc should be added with a time interval of 2mins between tubes, so do the measurements procedure.

7. 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 and aTc. Repeat measurement several hours a time until OD600 reach to 0.8,it will take about 7hours in average.