Signalling

Part:BBa_K176026

Designed by: Danqian Liu, Chao Li, Hao Jiang   Group: iGEM09_USTC   (2009-07-16)

TetR repressible AHL->GFP Receiver (Very High LuxR): pCon 0.70->luxR+pLux/Tet->GFP

AHL -> GFP

TetR repressible

Very high LuxR level

K176028 with 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
  • 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 985
    Illegal BsaI.rc site found at 1729

MEASUREMENT

=measurement1

We mesured the hybrid promoter's response to AHL concentration through different devices.
INPUT: AHL
DEVICE: 
             TetR repressible AHL->GFP Receiver:
             K176026 ( pCon 0.70->luxR+pLux/Tet->GFP );
             K176126 ( pCon 0.36->luxR+pLux/Tet->GFP );
             K176128 ( pCon 0.15->luxR+pLux/Tet->GFP );
             K176130 ( pCon 0.04->luxR+pLux/Tet->GFP );
OUTPUT: GFP

Model

To see the details of the model, click [http://2009.igem.org/Team:USTC/Modeling/Model-2 here] please.

data

K176026

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

026.jpg

protocol

AHLHybrid 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.

Measurement2

  • K176026([[1]]) is a device which can response to different concentrations of AHL. Here, we use K176126 as a detector to examing the AHL concentrations produced by constitutive promoters + luxI.
constitutive promoters + luxI-LVA: 
                                                                K176020( J23101->luxI-LVA);
                                                                K176021( J23107->luxI-LVA);
                                                                K176022( J23115->luxI-LVA);
                                                                K176023( J23109->luxI-LVA);
                                                                K176024( J23103->luxI-LVA);

measuring device: 
                                    K176217:( K176020 + K176026):( K176083 + GFP); 
                                    K176218:( K176021 + K176026):( K176084 + GFP);
                                    K176219:( K176022 + K176026):( K176085 + GFP);
                                    K176220:( K176023 + K176026):( K176086 + GFP);
                                    K176221:( K176024 + K176026):( K176087 + GFP);

data

K176217

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
0 0.0875 100.2605 1147.557098 4587.109158 401.3720513
60 0.120666667 148.52 1229.380903 4914.181969 592.9779576
120 0.145 198.92 1370.07619 5476.580687 794.1041996
180 0.207666667 292.4516667 1404.89787 5615.772756 1166.208809

K176218


T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
0 0.1035 75.11366667 721.4097924 2883.678268 298.4607008
60 0.150333333 123.0506667 808.6659075 3232.465553 485.9473215
120 0.1995 195.035 966.403576 3862.987472 770.6660008
180 0.308333333 351.1483333 1131.802978 4524.135501 1394.941779

K176219

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
0 0.2515 32.2705 125.5594805 501.8966322 126.227003
60 0.372833333 43.24566667 112.4353648 449.4358429 167.5646634
120 0.474833333 68.484 139.8281768 558.9326329 265.3998452
180 0.594833333 116.7865 192.791143 770.6405364 458.4026791

K176220

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
0 0.2485 7.723 32.1438897 128.4881869 31.92931443
60 0.4105 12.202 29.72814969 118.8317932 48.78045109
120 0.491666667 17.4355 36.20398381 144.7175273 71.15278427
180 0.611 31.26466667 51.84736322 207.2485239 126.6288481

K176221

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
0 0.219666667 11.49566667 52.33304675 209.1899378 45.95205634
60 0.3495 16.41366667 46.97848701 187.7862534 65.63129556
120 0.454833333 24.69433333 54.29412657 217.0289266 98.71199013
180 0.5765 43.4475 75.41615552 301.4596295 173.7914764 
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

Figure 1- the relationship chart between FLU and OD600 of diffrent constitutive promoters + luxI-LVA
Figure 2- The time response of K176126 to different constitutive promoters + luxI-LVA. We can see from the figure that the more strong the constitutive promoter is , the more strong response it shows.

protocol

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 1:100 into 3ml fresh medium and grown for 3hrs.

5. 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) every 30 minutes in the next 4hrs.


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Categories
Parameters
n/aTetR repressible AHL->GFP Receiver (Very High LuxR): pCon 0.70->luxR+pLux/Tet->GFP