Signalling

Part:BBa_K176126

Designed by: Danqian Liu, Zongxiao He, Hao Jiang   Group: iGEM09_USTC   (2009-09-22)
Revision as of 15:13, 20 October 2009 by Dqliu (Talk | contribs) (→‎plot)

TetR repressible AHL->GFP Receiver (High LuxR): pCon 0.36->luxR+pLux/Tet->GFP

AHL -> GFP Receiver

TetR repressible

High LuxR level

K176125 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 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

data

K176126

T(hour) OD600 FLU(arbitrary) FLU/OD(arbitrary) AHL ST.FLU/OD(ustc_st1)
0.5 0.147 0.724 4.925170068 0 19.68729291
0.5 0.149666667 2.844 19.00222717 1.00E-10 75.95725775
0.5 0.157 9.368666667 59.67303609 1.00E-09 238.529944
0.5 0.152 11.27633333 74.18640351 1.00E-08 296.5439641
0.5 0.149333333 11.57933333 77.54017857 1.00E-07 309.9499483
0.5 0.147 12.41233333 84.43764172 1.00E-05 337.5210526
1.5 0.268 1.283666667 4.789800995 0 19.14618457
1.5 0.276666667 3.247666667 11.73855422 1.00E-10 46.9223097
1.5 0.282666667 38.829 137.3667453 1.00E-09 549.0935975
1.5 0.270333333 94.57733333 349.8545006 1.00E-08 1398.467045
1.5 0.255666667 92.93366667 363.4954368 1.00E-07 1452.993711
1.5 0.232 75.93966667 327.3261494 1.00E-05 1308.414876
2.5 0.381666667 2.555 6.694323144 0 26.75909639
2.5 0.439 5.467 12.45330296 1.00E-10 49.77936188
2.5 0.437333333 92.855 212.3208841 1.00E-09 848.7064162
2.5 0.409333333 227.38 555.4885993 1.00E-08 2220.444495
2.5 0.382 222.79 583.2198953 1.00E-07 2331.294301
2.5 0.299333333 163.65 546.714922 1.00E-05 2185.373634
3.5 0.584 4.336 7.424657534 0 29.67844879
3.5 0.611 7.840333333 12.83196945 1.00E-10 51.29299856
3.5 0.599333333 154.5333333 257.8420467 1.00E-09 1030.667333
3.5 0.574333333 412.9266667 718.9669182 1.00E-08 2873.913412
3.5 0.518333333 401.8466667 775.266881 1.00E-07 3098.960231
3.5 0.381666667 272.7966667 714.7510917 1.00E-05 2857.061565 
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

126.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 (High LuxR): pCon 0.36->luxR+pLux/Tet->GFP