Signalling

Part:BBa_K176026

Designed by: Danqian Liu, Chao Li, Hao Jiang   Group: iGEM09_USTC   (2009-07-16)
Revision as of 07:56, 20 October 2009 by Dqliu (Talk | contribs) (→‎data)

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

We mesured the hybrid promoter's response to AHL concentration through different device.
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

data

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.


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