Signalling

Part:BBa_K176126

Designed by: Danqian Liu, Zongxiao He, Hao Jiang   Group: iGEM09_USTC   (2009-09-22)
Revision as of 01:49, 21 October 2009 by Dqliu (Talk | contribs)

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

MEASUREMENT1

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.

MEARSUREMENT2

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

data

K176136

AHL(M) T(h) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1)
0 0.5 0.191333333 0.230333333 1.203832753 4.811481825
1.00E-10 0.5 0.191 0.313666667 1.642233857 6.56368448
1.00E-09 0.5 0.193666667 0.412 2.127366609 8.502664306
1.00E-08 0.5 0.189333333 0.464333333 2.452464789 9.802017541
1.00E-07 0.5 0.188 0.483 2.569148936 10.26838104
1.00E-05 0.5 0.161666667 0.575 3.556701031 14.21543178
0 1.5 0.249 0.286666667 1.151271754 4.60140589
1.00E-10 1.5 0.249333333 0.411666667 1.651069519 6.598998876
1.00E-09 1.5 0.250666667 1.536 6.127659574 24.49104546
1.00E-08 1.5 0.248 2.437666667 9.829301075 39.28577568
1.00E-07 1.5 0.244 2.441666667 10.0068306 39.99532614
1.00E-05 1.5 0.202666667 2.552333333 12.59375 50.33473221
0 3 0.356666667 1.029333333 2.885981308 11.53469748
1.00E-10 3 0.361666667 1.441333333 3.985253456 15.92827121
1.00E-09 3 0.357333333 7.754333333 21.7005597 86.73285252
1.00E-08 3 0.356666667 11.61666667 32.57009346 130.1762328
1.00E-07 3 0.354 11.41833333 32.25517891 128.9175816
1.00E-05 3 0.297333333 11.67633333 39.27017937 156.9551534

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

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 1- this steregram shows the response of K176136 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 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 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.



[edit]
Categories
Parameters
n/aTetR repressible AHL->GFP Receiver (High LuxR): pCon 0.36->luxR+pLux/Tet->GFP