Device

Part:BBa_K176140

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

aTc&AHL->GFP: pCon 0.15->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

K176140

AHL(M) T(h) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1)
0 0.5 0.236666667 2.070666667 8.749295775 34.96920773
1.00E-10 0.5 0.237666667 1.967 8.276297335 33.07872636
1.00E-09 0.5 0.246 4.862666667 19.76693767 79.00454704
1.00E-08 0.5 0.242666667 4.767 19.64423077 78.51411179
1.00E-07 0.5 0.237666667 5.182 21.80364656 87.14487036
1.00E-05 0.5 0.24 5.683 23.67916667 94.6409539
0 1.5 0.417666667 2.472 5.918595371 23.65545712
1.00E-10 1.5 0.415 3.980666667 9.591967871 38.33720172
1.00E-09 1.5 0.413333333 149.79 362.3951613 1448.421908
1.00E-08 1.5 0.405333333 132.8333333 327.7138158 1309.807417
1.00E-07 1.5 0.402 136.3766667 339.2454395 1355.89704
1.00E-05 1.5 0.396 134.04 338.4848485 1352.857108
0 3 0.616 6.812 11.05844156 44.19840751
1.00E-10 3 0.651666667 11.56666667 17.74936061 70.9406899
1.00E-09 3 0.616 635.81 1032.159091 4125.336095
1.00E-08 3 0.608 646.3333333 1063.048246 4248.793947
1.00E-07 3 0.605 655.2066667 1082.986226 4328.482118
1.00E-05 3 0.594 696.75 1172.979798 4688.168657

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

plot

Figure 2- this steregram shows the response of K176138 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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