Device

Part:BBa_K176030

Designed by: Chao Li, Danqian Liu, Hao Jiang   Group: iGEM09_USTC   (2009-07-20)
Revision as of 17:33, 20 October 2009 by Dqliu (Talk | contribs)

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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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 855
    Illegal NheI site found at 878
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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

Figure 1- the hybrid promoter's ( K176000) response to aTc and AHL at definit time( here we choose t=7h) by examing K176071
Figure 2- the hybrid promoter's (K176000) time response at different conditions by examing K176071. The 11 different conditions , whose details are shown in the chart below, differs from each other with different AHL concentration and aTc concentration.
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

Figure 1- the hybrid promoter's ( K176000) response to aTc and AHL at definit time( here we choose t=7h) by examing K176075
Figure 2- the hybrid promoter's (K176000) time response at different conditions by examing K176075. The 11 different conditions , whose details are shown in the chart below, differs from each other with different AHL concentration and aTc concentration.
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,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.


[edit]
Categories
Parameters
n/aRepressible AHL->GFP Receiver: PoPS->tetR-LVA+pCon 0.70->luxR+pLux/Tet->GFP