Device

Part:BBa_K176029

Designed by: Chao Li, Danqian Liu, Hao Jiang   Group: iGEM09_USTC   (2009-07-20)

Repressible AHL->GFP Receiver: PoPS->tetR+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 797
    Illegal NheI site found at 820
  • 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 1775
    Illegal BsaI.rc site found at 2519


MEASUREMENT

By ligating the device( K176029 ) after some definite constitutive promoter, we can exam the response of the hybrid promoter to both 
AHL and aTc. Here the tetR part is attached LVA.
INPUT : AHL, aTc
DEVICE: K176078( J23109 + K176029)
OUTPUT: GFP

data

K176078

T(min) NUMBER AHL(M) ATC(ng/ml) FLU(arbitrary) OD600 FLU/OD600 ST.FLU/OD600(ustc_st1)
0 1 0.00E+00 0 0.094 0.087 1.08045977 4.318902227
0 2 1.00E-06 0 0.112 0.086333333 1.297297297 5.185662938
0 3 1.00E-06 0.001 0.060333333 0.090666667 0.665441176 2.659955936
0 4 1.00E-06 0.01 0.087 0.094666667 0.919014085 3.673558318
0 5 1.00E-06 0.1 0.022666667 0.099 0.228956229 0.915202578
0 6 1.00E-06 1 0.038666667 0.099666667 0.387959866 1.550784931
0 7 1.00E-04 0 0.211333333 0.107333333 1.968944099 7.870424509
0 8 1.00E-04 0.001 0.120666667 0.108 1.117283951 4.466098855
0 9 1.00E-04 0.01 0.057666667 0.107 0.53894081 2.154298317
0 10 1.00E-04 0.1 0.044 0.106 0.41509434 1.659249069
0 11 1.00E-04 1 0.024 0.109 0.220183486 0.880135453
120 1 0.00E+00 0 0.930666667 0.263 3.538656527 14.1450075
120 2 1.00E-06 0 0.910333333 0.282666667 3.220518868 12.87332161
120 3 1.00E-06 0.001 91.18233333 0.268666667 339.3883375 1356.630841
120 4 1.00E-06 0.01 102.1966667 0.282333333 361.9716647 1446.902765
120 5 1.00E-06 0.1 87.97266667 0.267666667 328.6650062 1313.766664
120 6 1.00E-06 1 6.056666667 0.106333333 56.95924765 227.6821667
120 7 1.00E-04 0 5.871333333 0.282666667 20.77122642 83.02844632
120 8 1.00E-04 0.001 140.8833333 0.27 521.7901235 2085.742189
120 9 1.00E-04 0.01 153.5733333 0.261 588.403576 2352.014934
120 10 1.00E-04 0.1 148.3833333 0.256 579.6223958 2316.914082
120 11 1.00E-04 1 23.95366667 0.123333333 194.2189189 776.3477592
300 1 0.00E+00 0 3.509666667 1.016 3.454396325 13.80819573
300 2 1.00E-06 0 7.015333333 0.937 7.487015297 29.92771035
300 3 1.00E-06 0.001 501.98 0.872666667 575.2253629 2299.337902
300 4 1.00E-06 0.01 632.6 0.921 686.8621064 2745.58143
300 5 1.00E-06 0.1 543.6 0.839333333 647.6568705 2588.867053
300 6 1.00E-06 1 60.689 0.178333333 340.3121495 1360.323578
300 7 1.00E-04 0 29.55633333 0.878 33.66324981 134.5614974
300 8 1.00E-04 0.001 910.6966667 0.845 1077.747535 4308.060657
300 9 1.00E-04 0.01 1009 0.833333333 1210.8 4839.908862
300 10 1.00E-04 0.1 1013.7 0.826666667 1226.25 4901.666867
300 11 1.00E-04 1 171.3966667 0.188 911.6843972 3644.259492 
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=5h)
Figure 2- the hybrid promoter's (K176000) time response at different conditions. 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
None