Device

Part:BBa_K176144

Designed by: Danqian Liu, Zongxiao He, Hao Jiang   Group: iGEM09_USTC   (2009-10-07)

aTc&AHL->GFP: pCon 0.01->tetR-LVA+pCon 0.36->luxR+pLux/Tet->GFP

Input aTc activates 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 7
    Illegal NheI site found at 30
    Illegal NheI site found at 898
    Illegal NheI site found at 921
  • 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 1876
    Illegal BsaI.rc site found at 2620


MEARSUREMENT

We test the response of the hybrid promoter( K176000) to both tetR and AHL. Here LuxR is ligated after J23107. 

INPUT : constitutive promoters +tetR , AHL
DEVICE: K176136( J23101 + K176126);
K176140( J23115 + K176126);
K176144( J23103 + K176126);
OUTPUT: GFP

Model

To see the details of the model, click [http://2009.igem.org/Team:USTC/Modeling/Model-3 here] please.

data

K176144

AHL(M) T(h) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1)
0 0.5 0.210333333 4.745 22.55942948 90.16558544
1.00E-10 0.5 0.245 7.730333333 31.55238095 126.1086369
1.00E-09 0.5 0.246333333 11.12366667 45.15696888 180.4834887
1.00E-08 0.5 0.25 11.69366667 46.77466667 186.9491074
1.00E-07 0.5 0.256666667 13.35266667 52.02337662 207.9271648
1.00E-05 0.5 0.249 14.77833333 59.35073628 237.2131746
0 1.5 0.430666667 10.12166667 23.50232198 93.93414061
1.00E-10 1.5 0.449 16.77366667 37.35783222 149.3118794
1.00E-09 1.5 0.441333333 133.0933333 301.570997 1205.319732
1.00E-08 1.5 0.429666667 189 439.8758728 1758.097013
1.00E-07 1.5 0.408333333 168.8066667 413.4040816 1652.294491
1.00E-05 1.5 0.38 138.9433333 365.6403509 1461.39229
0 3 0.666 20.16933333 30.28428428 121.0403049
1.00E-10 3 0.676 37.01733333 54.75936884 218.8623854
1.00E-09 3 0.648666667 451.2633333 695.6783145 2780.488867
1.00E-08 3 0.638333333 774.2666667 1212.950392 4847.923228
1.00E-07 3 0.561666667 580.5466667 1033.614243 4131.152052
1.00E-05 3 0.464333333 360.73 776.8772434 3105.024953

For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

Figure 3- this steregram shows the response of K176144 to both time and AHL. From this gram, we can see that the GFP expression reaches its top at some definite AHL(1.E-08) concentration at any time in the range of allowable error.
Figure 4- the steregram shows the response of different device( K176136; K176140; K176144 ) to both tetR and AHL. In this gram, the score( click here to see the details) represents the relative strength of different promoter before tetR-LVA, that is , to represent the INPUT of tetR.

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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Parameters
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