Template:USTC//K176026
Contents
MEASUREMENT
We mesured the hybrid promoter's response to AHL concentration through different devices.
INPUT: AHL
DEVICE:
TetR repressible AHL->GFP Receiver:
K176028 ( pCon 0.70->luxR+pLux/Tet)->GFP ;
K176125 ( pCon 0.36->luxR+pLux/Tet)->GFP ;
K176127 ( pCon 0.15->luxR+pLux/Tet)->GFP ;
K176129 (pCon 0.04->luxR+pLux/Tet)->GFP ;
OUTPUT: GFP
data
K176026 ( K176028 + GFP)
| T(hour) | OD600 | FLU(arbitrary) | FLU/OD(arbitrary) | AHL(M) | ST.FLU/OD(ustc_st1) |
| 0.5 | 0.152 | 0.513666667 | 3.379385965 | 0 | 13.50835818 |
| 0.5 | 0.164 | 0.547333333 | 3.337398374 | 1.00E-10 | 13.34052194 |
| 0.5 | 0.177 | 0.550666667 | 3.111111111 | 1.00E-09 | 12.43598797 |
| 0.5 | 0.146 | 0.442333333 | 3.029680365 | 1.00E-08 | 12.11048633 |
| 0.5 | 0.166 | 0.563666667 | 3.395582329 | 1.00E-07 | 13.57309961 |
| 0.5 | 0.167333333 | 0.541333333 | 3.235059761 | 1.00E-05 | 12.93144566 |
| 1.5 | 0.247333333 | 0.810666667 | 3.277628032 | 0 | 13.10160304 |
| 1.5 | 0.252 | 1.463 | 5.805555556 | 1.00E-10 | 23.20644184 |
| 1.5 | 0.254 | 15.52866667 | 61.13648294 | 1.00E-09 | 244.3797535 |
| 1.5 | 0.245 | 34.521 | 140.9020408 | 1.00E-08 | 563.2251701 |
| 1.5 | 0.261 | 35.881 | 137.4750958 | 1.00E-07 | 549.526705 |
| 1.5 | 0.254 | 39.50733333 | 155.5406824 | 1.00E-05 | 621.7399465 |
| 2.5 | 0.372 | 1.536666667 | 4.130824373 | 0 | 16.51206928 |
| 2.5 | 0.405333333 | 3.574666667 | 8.819078947 | 1.00E-10 | 35.2523442 |
| 2.5 | 0.411666667 | 68.232 | 165.745749 | 1.00E-09 | 662.5324739 |
| 2.5 | 0.403 | 177.9833333 | 441.6459884 | 1.00E-08 | 1765.383493 |
| 2.5 | 0.383666667 | 187.4133333 | 488.479583 | 1.00E-07 | 1952.59057 |
| 2.5 | 0.404 | 192.4866667 | 476.4521452 | 1.00E-05 | 1904.513512 |
| 3.5 | 0.722 | 3.111666667 | 4.309787627 | 0 | 17.22743585 |
| 3.5 | 0.873333333 | 8.359333333 | 9.571755725 | 1.00E-10 | 38.26100542 |
| 3.5 | 0.781 | 175.7666667 | 225.0533504 | 1.00E-09 | 899.6016725 |
| 3.5 | 0.734333333 | 373.0766667 | 508.0481162 | 1.00E-08 | 2030.811513 |
| 3.5 | 0.674333333 | 396.2633333 | 587.6371725 | 1.00E-07 | 2348.951403 |
| 3.5 | 0.72 | 400.7966667 | 556.662037 | 1.00E-05 | 2225.135056 |
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.
plot
protocol
AHLHybrid 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.