Difference between revisions of "Part:BBa K176125"
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K176125 SequenceAndFeatures</partinfo> | <partinfo>BBa_K176125 SequenceAndFeatures</partinfo> | ||
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Latest revision as of 08:15, 20 October 2009
TetR repressible AHL->PoPS Receiver (High LuxR): pCon 0.36->luxR+pLux/Tet->PoPS
AHL -> PoPS Receiver
TetR repressible
High LuxR level
Similar to F2620, but use pLux/Tet hybrid promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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
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
protocol
AHLHybrid 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.