Signalling

Part:BBa_K176127

Designed by: Danqian Liu, Zongxiao He, Hao Jiang   Group: iGEM09_USTC   (2009-09-22)

TetR repressible AHL->PoPS Receiver (Medium LuxR): pCon 0.15->luxR+pLux/Tet->PoPS

AHL -> PoPS Receiver

TetR repressible

Medium LuxR level

Similar to F2620, but use pLux/Tet hybrid promoter.

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

MEASUREMENT

We mesured the hybrid promoter's response to AHL concentration through different devices.
INPUT: AHL
DEVICE: 
             TetR repressible AHL->GFP Receiver:
              K176028 ( pCon 0.70->luxR+pLux/Tet)->GFP ;
             K176125 ( pCon 0.36->luxR+pLux/Tet)->GFP ;
             K176127 ( pCon 0.15->luxR+pLux/Tet)->GFP ;
             K176129 (pCon 0.04->luxR+pLux/Tet)->GFP ;
OUTPUT: GFP

data

K176128

T(hour) OD600 FLU(arbitrary) FLU/OD(arbitrary) AHL ST.FLU/OD(ustc_st1)
0.5 0.144 0.805 5.590277778 0 22.34591589
0.5 0.156 1.197333333 7.675213675 1.00E-10 30.67999231
0.5 0.160333333 4.548333333 28.36798337 1.00E-09 113.394825
0.5 0.16 7.007 43.79375 1.00E-08 175.0559619
0.5 0.161333333 8.152333333 50.53099174 1.00E-07 201.986616
0.5 0.157333333 8.976666667 57.05508475 1.00E-05 228.0652546
1.5 0.256666667 1.568 6.109090909 0 24.4197582
1.5 0.274 1.970333333 7.190997567 1.00E-10 28.74444405
1.5 0.282666667 9.054 32.03066038 1.00E-09 128.0355773
1.5 0.276333333 64.781 234.4306393 1.00E-08 937.0853393
1.5 0.274333333 89.16466667 325.0230863 1.00E-07 1299.208883
1.5 0.264333333 83.70166667 316.6519546 1.00E-05 1265.74711
2.5 0.356 2.742333333 7.703183521 0 30.79179566
2.5 0.391666667 3.256666667 8.314893617 1.00E-10 33.23697333
2.5 0.421333333 17.24066667 40.9193038 1.00E-09 163.5659903
2.5 0.396666667 169.93 428.394958 1.00E-08 1712.415389
2.5 0.391666667 271.61 693.4723404 1.00E-07 2772.004399
2.5 0.371 247.67 667.574124 1.00E-05 2668.481928
3.5 0.471666667 4.409 9.34770318 0 37.36540425
3.5 0.499 5.094 10.20841683 1.00E-10 40.80591931
3.5 0.557333333 28.99833333 52.03050239 1.00E-09 207.9805828
3.5 0.518 282.62 545.5984556 1.00E-08 2180.910803
3.5 0.514333333 463.9933333 902.1257291 1.00E-07 3606.050802
3.5 0.483333333 470.7433333 973.9517241 1.00E-05 3893.159548
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

K176128.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.


[edit]
Categories
Parameters
n/aTetR repressible AHL->PoPS Receiver (Medium LuxR): pCon 0.15->luxR+pLux/Tet->PoPS