Difference between revisions of "Part:BBa K176126"

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=='''MEASUREMENT'''==
 
=='''MEASUREMENT'''==
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===MEASUREMENT1===
 
  '''We mesured the hybrid promoter's response to AHL concentration through different devices.'''
 
  '''We mesured the hybrid promoter's response to AHL concentration through different devices.'''
 
  INPUT: AHL
 
  INPUT: AHL
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  OUTPUT: GFP
 
  OUTPUT: GFP
  
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===data===
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====data====
 
'''K176126'''
 
'''K176126'''
 
{| {{table}}
 
{| {{table}}
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  For the details of the definition of '''ST.FLU''' and the unit '''ustc_st1''', please click [[here]].  
 
  For the details of the definition of '''ST.FLU''' and the unit '''ustc_st1''', please click [[here]].  
  
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===plot===
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====plot====
  
 
[[Image:126.jpg|center]]
 
[[Image:126.jpg|center]]
  
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===protocol===
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====protocol====
 
AHLHybrid promoter:BBa_K176026, BBa_K176126, BBa_K176128, BBa_K176130
 
AHLHybrid promoter:BBa_K176026, BBa_K176126, BBa_K176128, BBa_K176130
  
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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.
 
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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===MEARSUREMENT2===
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{{USTC//measure-126}}
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{{USTC//K176136&section=1}}
  
  

Revision as of 01:43, 21 October 2009

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

Template:USTC//K176136&section=1