Difference between revisions of "Part:BBa K176030"
Line 14: | Line 14: | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K176030 SequenceAndFeatures</partinfo> | <partinfo>BBa_K176030 SequenceAndFeatures</partinfo> | ||
+ | |||
+ | {{USTC//measure-30}} | ||
+ | |||
+ | **'''K176071''' | ||
+ | {{USTC//K176071}} | ||
+ | |||
+ | **'''K176075''' | ||
+ | {{USTC//K176075}} | ||
+ | |||
+ | {{USTC//protocol-AHL+aTc}} | ||
Latest revision as of 17:33, 20 October 2009
Repressible AHL->GFP Receiver: PoPS->tetR-LVA+pCon 0.70->luxR+pLux/Tet->GFP
Repressible AHL->GFP Receiver
Input PoPS represses GFP.
Input AHL activates GFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 855
Illegal NheI site found at 878 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1833
Illegal BsaI.rc site found at 2577
MEASUREMENT
We tested the response of the hybrid promoter (K176000) to both AHL and aTc.
INPUT : AHL, aTc DEVICE: K176071( J23107 + K176030); K176075( J23115 + K176030); OUTPUT: GFP
- K176071
data
K176071
T(min) | NUMBER | AHL(M) | ATC(M) | FLU(arbitrary) | OD600 | FLU/OD600 | ST.FLU/OD600(ustc_st1) |
0 | 1 | 0.00E+00 | 0 | 0.011 | 0.467 | 42.45454545 | 169.7027839 |
0 | 2 | 1.00E-06 | 0 | 0.020666667 | 1.170666667 | 56.64516129 | 226.426675 |
0 | 3 | 1.00E-06 | 0.001 | 0.016 | 0.304 | 19 | 75.94835512 |
0 | 4 | 1.00E-06 | 0.01 | 0.016 | 0.236666667 | 14.79166667 | 59.12646067 |
0 | 5 | 1.00E-06 | 0.1 | 0.019666667 | 0.461333333 | 23.45762712 | 93.76674709 |
0 | 6 | 1.00E-06 | 1 | 0.022 | 0.267333333 | 12.15151515 | 48.57303095 |
0 | 7 | 1.00E-04 | 0 | 0.02 | 0.193333333 | 9.666666667 | 38.6403912 |
0 | 8 | 1.00E-04 | 0.001 | 0.017 | 0.147666667 | 8.68627451 | 34.72148743 |
0 | 9 | 1.00E-04 | 0.01 | 0.02 | 0.077333333 | 3.866666667 | 15.45615648 |
0 | 10 | 1.00E-04 | 0.1 | 0.021 | 0.133333333 | 6.349206349 | 25.37956729 |
0 | 11 | 1.00E-04 | 1 | 0.021 | 0.103666667 | 4.936507937 | 19.73261357 |
60 | 1 | 0.00E+00 | 0 | 0.028666667 | 0.116 | 4.046511628 | 16.17504748 |
60 | 2 | 1.00E-06 | 0 | 0.031 | 0.078 | 2.516129032 | 10.05767691 |
60 | 3 | 1.00E-06 | 0.001 | 0.033 | 0.251666667 | 7.626262626 | 30.48432117 |
60 | 4 | 1.00E-06 | 0.01 | 0.036 | 3.021 | 83.91666667 | 335.4385684 |
60 | 5 | 1.00E-06 | 0.1 | 0.037 | 4.382 | 118.4324324 | 473.4078124 |
60 | 6 | 1.00E-06 | 1 | 0.033 | 0.552666667 | 16.74747475 | 66.94437681 |
60 | 7 | 1.00E-04 | 0 | 0.038 | 0.210333333 | 5.535087719 | 22.12530567 |
60 | 8 | 1.00E-04 | 0.001 | 0.037 | 0.625333333 | 16.9009009 | 67.55766439 |
60 | 9 | 1.00E-04 | 0.01 | 0.037666667 | 4.063 | 107.8672566 | 431.175827 |
60 | 10 | 1.00E-04 | 0.1 | 0.036 | 6.538333333 | 181.6203704 | 725.9878098 |
60 | 11 | 1.00E-04 | 1 | 0.027 | 0.766 | 28.37037037 | 113.4043665 |
180 | 1 | 0.00E+00 | 0 | 0.079333333 | 0.127666667 | 1.609243697 | 6.432600621 |
180 | 2 | 1.00E-06 | 0 | 0.082 | 0.173333333 | 2.113821138 | 8.449538866 |
180 | 3 | 1.00E-06 | 0.001 | 0.091666667 | 1.005333333 | 10.96727273 | 43.8392802 |
180 | 4 | 1.00E-06 | 0.01 | 0.077 | 25.80233333 | 335.0952381 | 1339.470113 |
180 | 5 | 1.00E-06 | 0.1 | 0.081 | 28.226 | 348.4691358 | 1392.929351 |
180 | 6 | 1.00E-06 | 1 | 0.027333333 | 3.880333333 | 141.9634146 | 567.4677804 |
180 | 7 | 1.00E-04 | 0 | 0.093 | 1.224 | 13.16129032 | 52.60938691 |
180 | 8 | 1.00E-04 | 0.001 | 0.092 | 8.581333333 | 93.27536232 | 372.8479127 |
180 | 9 | 1.00E-04 | 0.01 | 0.081 | 37.95933333 | 468.6337449 | 1873.261162 |
180 | 10 | 1.00E-04 | 0.1 | 0.07 | 39.35 | 562.1428571 | 2247.043439 |
180 | 11 | 1.00E-04 | 1 | 0.020333333 | 3.697 | 181.8196721 | 726.7844751 |
420 | 1 | 0.00E+00 | 0 | 0.537666667 | 0.515 | 0.957842529 | 3.828766557 |
420 | 2 | 1.00E-06 | 0 | 0.6 | 0.521 | 0.868333333 | 3.470973072 |
420 | 3 | 1.00E-06 | 0.001 | 0.613 | 1.356333333 | 2.212615552 | 8.844447983 |
420 | 4 | 1.00E-06 | 0.01 | 0.53 | 58.196 | 109.8037736 | 438.916631 |
420 | 5 | 1.00E-06 | 0.1 | 0.479333333 | 219.83 | 458.6161335 | 1833.217946 |
420 | 6 | 1.00E-06 | 1 | 0.058333333 | 12.569 | 215.4685714 | 861.2886095 |
420 | 7 | 1.00E-04 | 0 | 0.604666667 | 5.103333333 | 8.439911797 | 33.73670623 |
420 | 8 | 1.00E-04 | 0.001 | 0.584 | 20.67833333 | 35.40810502 | 141.5361755 |
420 | 9 | 1.00E-04 | 0.01 | 0.440333333 | 335.6466667 | 762.2558668 | 3046.95154 |
420 | 10 | 1.00E-04 | 0.1 | 0.331666667 | 295.83 | 891.9497487 | 3565.37454 |
420 | 11 | 1.00E-04 | 1 | 0.043 | 18.108 | 421.1162791 | 1683.320458 |
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.
plot
conditions | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
AHL/M | 0.00E+00 | 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 |
Atc/(ng/ml) | 0 | 0 | 2 | 20 | 200 | 2000 | 0 | 2 | 20 | 200 | 2000 |
- K176075
data
K176075
T(min) | NUMBER | AHL(M) | ATC(M) | FLU(arbitrary) | OD600 | FLU/OD600 | ST.FLU/OD600(ustc_st1) |
0 | 1 | 0.00E+00 | 0 | 0.029333333 | 0.798666667 | 27.22727273 | 108.8350831 |
0 | 2 | 1.00E-06 | 0 | 0.03 | 0.480666667 | 16.02222222 | 64.04533806 |
0 | 3 | 1.00E-06 | 0.001 | 0.033 | 0.461666667 | 13.98989899 | 55.92156929 |
0 | 4 | 1.00E-06 | 0.01 | 0.030333333 | 0.227333333 | 7.494505495 | 29.95765078 |
0 | 5 | 1.00E-06 | 0.1 | 0.031 | 0.197333333 | 6.365591398 | 25.44506295 |
0 | 6 | 1.00E-06 | 1 | 0.036 | 0.417 | 11.58333333 | 46.30184808 |
0 | 7 | 1.00E-04 | 0 | 0.032666667 | 0.338666667 | 10.36734694 | 41.44120773 |
0 | 8 | 1.00E-04 | 0.001 | 0.036 | 0.996666667 | 27.68518519 | 110.6654882 |
0 | 9 | 1.00E-04 | 0.01 | 0.038 | 1.183 | 31.13157895 | 124.4416954 |
0 | 10 | 1.00E-04 | 0.1 | 0.035 | 0.587333333 | 16.78095238 | 67.07819635 |
0 | 11 | 1.00E-04 | 1 | 0.03 | 0.521333333 | 17.37777778 | 69.46387568 |
60 | 1 | 0.00E+00 | 0 | 0.059 | 0.326666667 | 5.536723164 | 22.131843 |
60 | 2 | 1.00E-06 | 0 | 0.069333333 | 9.875666667 | 142.4375 | 569.3628333 |
60 | 3 | 1.00E-06 | 0.001 | 0.062 | 16.431 | 265.016129 | 1059.344162 |
60 | 4 | 1.00E-06 | 0.01 | 0.059 | 16.61166667 | 281.5536723 | 1125.449384 |
60 | 5 | 1.00E-06 | 0.1 | 0.063 | 15.81466667 | 251.026455 | 1003.423492 |
60 | 6 | 1.00E-06 | 1 | 0.039666667 | 0.623 | 15.70588235 | 62.78083844 |
60 | 7 | 1.00E-04 | 0 | 0.062 | 22.895 | 369.2741935 | 1476.093031 |
60 | 8 | 1.00E-04 | 0.001 | 0.064 | 24.39466667 | 381.1666667 | 1523.630598 |
60 | 9 | 1.00E-04 | 0.01 | 0.069 | 24.72833333 | 358.3816425 | 1432.552434 |
60 | 10 | 1.00E-04 | 0.1 | 0.063 | 21.76166667 | 345.4232804 | 1380.754209 |
60 | 11 | 1.00E-04 | 1 | 0.057 | 3.875333333 | 67.98830409 | 271.7684139 |
150 | 1 | 0.00E+00 | 0 | 0.154666667 | 0.936333333 | 6.05387931 | 24.19906188 |
150 | 2 | 1.00E-06 | 0 | 0.148 | 43.15633333 | 291.5968468 | 1165.594783 |
150 | 3 | 1.00E-06 | 0.001 | 0.133 | 60.345 | 453.7218045 | 1813.653933 |
150 | 4 | 1.00E-06 | 0.01 | 0.131 | 59.66 | 455.4198473 | 1820.441489 |
150 | 5 | 1.00E-06 | 0.1 | 0.124333333 | 54.89533333 | 441.5174263 | 1764.869594 |
150 | 6 | 1.00E-06 | 1 | 0.038 | 0.9 | 23.68421053 | 94.67246483 |
150 | 7 | 1.00E-04 | 0 | 0.117 | 82.428 | 704.5128205 | 2816.136309 |
150 | 8 | 1.00E-04 | 0.001 | 0.113333333 | 76.71966667 | 676.9382353 | 2705.91292 |
150 | 9 | 1.00E-04 | 0.01 | 0.113 | 74.73166667 | 661.3421829 | 2643.571103 |
150 | 10 | 1.00E-04 | 0.1 | 0.107666667 | 67.21 | 624.2414861 | 2495.269161 |
150 | 11 | 1.00E-04 | 1 | 0.04 | 4.403333333 | 110.0833333 | 440.0341101 |
420 | 1 | 0.00E+00 | 0 | 0.744 | 11.972 | 16.09139785 | 64.32185254 |
420 | 2 | 1.00E-06 | 0 | 0.783333333 | 379.65 | 484.6595745 | 1937.32092 |
420 | 3 | 1.00E-06 | 0.001 | 0.746 | 512.8733333 | 687.4977659 | 2748.12234 |
420 | 4 | 1.00E-06 | 0.01 | 0.733 | 512.4566667 | 699.1223283 | 2794.588993 |
420 | 5 | 1.00E-06 | 0.1 | 0.722666667 | 488.05 | 675.345941 | 2699.548071 |
420 | 6 | 1.00E-06 | 1 | 0.080666667 | 3.055333333 | 37.87603306 | 151.4011794 |
420 | 7 | 1.00E-04 | 0 | 0.583666667 | 712.9066667 | 1221.427756 | 4882.390996 |
420 | 8 | 1.00E-04 | 0.001 | 0.513666667 | 624.8533333 | 1216.456846 | 4862.520871 |
420 | 9 | 1.00E-04 | 0.01 | 0.470666667 | 562.26 | 1194.603399 | 4775.166485 |
420 | 10 | 1.00E-04 | 0.1 | 0.430333333 | 523.6433333 | 1216.831913 | 4864.020119 |
420 | 11 | 1.00E-04 | 1 | 0.077 | 51.26766667 | 665.8138528 | 2661.445628 |
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.
plot
conditions | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
AHL/M | 0.00E+00 | 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 |
Atc/(ng/ml) | 0 | 0 | 2 | 20 | 200 | 2000 | 0 | 2 | 20 | 200 | 2000 |
protocols
aTc,AHLHybrid 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.