Device

Part:BBa_K176075

Designed by: Zongxiao He, Hao Jiang   Group: iGEM09_USTC   (2009-07-30)

aTc&AHL->GFP: pCon 0.15->tetR-LVA+pCon 0.70->luxR+pLux/Tet->GFP

Input aTc activates 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 7
    Illegal NheI site found at 30
    Illegal NheI site found at 898
    Illegal NheI site found at 921
  • 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 1876
    Illegal BsaI.rc site found at 2620

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

Model

To see the details of the model, click [http://2009.igem.org/Team:USTC/Modeling/Model-3 here] please.

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.


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Parameters
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