Difference between revisions of "Part:BBa K176126"
Line 16: Line 16:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K176126 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K176126 SequenceAndFeatures</partinfo>
 +
 +
=='''MEASUREMENT'''==
 +
'''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'''
 +
{| {{table}}
 +
{|class="sortable" style="border:3px solid black; width: 70%" align="center"
 +
| align="center" style="background:#f0f0f0;"|'''T(hour)'''
 +
| align="center" style="background:#f0f0f0;"|'''OD600'''
 +
| align="center" style="background:#f0f0f0;"|'''FLU(arbitrary)'''
 +
| align="center" style="background:#f0f0f0;"|'''FLU/OD(arbitrary)'''
 +
| align="center" style="background:#f0f0f0;"|'''AHL'''
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| align="center" style="background:#f0f0f0;"|'''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===
 +
 +
[[Image:K176126.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.
 +
  
  

Revision as of 08:12, 20 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

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

K176126.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.