Difference between revisions of "Part:BBa K176128"
Line 16: Line 16:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K176128 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K176128 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===
 +
'''K176128'''
 +
 +
{| {{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'''
 +
| align="center" style="background:#f0f0f0;"|'''ST.FLU/OD(ustc_st1)'''
 +
|-
 +
| 0.5||0.144||0.805||5.590277778||0||22.34591589
 +
|-
 +
| 0.5||0.156||1.197333333||7.675213675||1.00E-10||30.67999231
 +
|-
 +
| 0.5||0.160333333||4.548333333||28.36798337||1.00E-09||113.394825
 +
|-
 +
| 0.5||0.16||7.007||43.79375||1.00E-08||175.0559619
 +
|-
 +
| 0.5||0.161333333||8.152333333||50.53099174||1.00E-07||201.986616
 +
|-
 +
| 0.5||0.157333333||8.976666667||57.05508475||1.00E-05||228.0652546
 +
|-
 +
| 1.5||0.256666667||1.568||6.109090909||0||24.4197582
 +
|-
 +
| 1.5||0.274||1.970333333||7.190997567||1.00E-10||28.74444405
 +
|-
 +
| 1.5||0.282666667||9.054||32.03066038||1.00E-09||128.0355773
 +
|-
 +
| 1.5||0.276333333||64.781||234.4306393||1.00E-08||937.0853393
 +
|-
 +
| 1.5||0.274333333||89.16466667||325.0230863||1.00E-07||1299.208883
 +
|-
 +
| 1.5||0.264333333||83.70166667||316.6519546||1.00E-05||1265.74711
 +
|-
 +
| 2.5||0.356||2.742333333||7.703183521||0||30.79179566
 +
|-
 +
| 2.5||0.391666667||3.256666667||8.314893617||1.00E-10||33.23697333
 +
|-
 +
| 2.5||0.421333333||17.24066667||40.9193038||1.00E-09||163.5659903
 +
|-
 +
| 2.5||0.396666667||169.93||428.394958||1.00E-08||1712.415389
 +
|-
 +
| 2.5||0.391666667||271.61||693.4723404||1.00E-07||2772.004399
 +
|-
 +
| 2.5||0.371||247.67||667.574124||1.00E-05||2668.481928
 +
|-
 +
| 3.5||0.471666667||4.409||9.34770318||0||37.36540425
 +
|-
 +
| 3.5||0.499||5.094||10.20841683||1.00E-10||40.80591931
 +
|-
 +
| 3.5||0.557333333||28.99833333||52.03050239||1.00E-09||207.9805828
 +
|-
 +
| 3.5||0.518||282.62||545.5984556||1.00E-08||2180.910803
 +
|-
 +
| 3.5||0.514333333||463.9933333||902.1257291||1.00E-07||3606.050802
 +
|-
 +
| 3.5||0.483333333||470.7433333||973.9517241||1.00E-05||3893.159548
 +
|}
 +
 +
For the details of the definition of '''ST.FLU''' and the unit '''ustc_st1''', please click [[here]].
 +
 +
===plot===
 +
 +
[[Image:K176128.jpg|center]]
 +
 +
===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 08:20, 20 October 2009

TetR repressible AHL->GFP Receiver (Medium LuxR): pCon 0.15->luxR+pLux/Tet->GFP

AHL -> GFP Receiver

TetR repressible

Medium LuxR level

K176127 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

K176128

T(hour) OD600 FLU(arbitrary) FLU/OD(arbitrary) AHL ST.FLU/OD(ustc_st1)
0.5 0.144 0.805 5.590277778 0 22.34591589
0.5 0.156 1.197333333 7.675213675 1.00E-10 30.67999231
0.5 0.160333333 4.548333333 28.36798337 1.00E-09 113.394825
0.5 0.16 7.007 43.79375 1.00E-08 175.0559619
0.5 0.161333333 8.152333333 50.53099174 1.00E-07 201.986616
0.5 0.157333333 8.976666667 57.05508475 1.00E-05 228.0652546
1.5 0.256666667 1.568 6.109090909 0 24.4197582
1.5 0.274 1.970333333 7.190997567 1.00E-10 28.74444405
1.5 0.282666667 9.054 32.03066038 1.00E-09 128.0355773
1.5 0.276333333 64.781 234.4306393 1.00E-08 937.0853393
1.5 0.274333333 89.16466667 325.0230863 1.00E-07 1299.208883
1.5 0.264333333 83.70166667 316.6519546 1.00E-05 1265.74711
2.5 0.356 2.742333333 7.703183521 0 30.79179566
2.5 0.391666667 3.256666667 8.314893617 1.00E-10 33.23697333
2.5 0.421333333 17.24066667 40.9193038 1.00E-09 163.5659903
2.5 0.396666667 169.93 428.394958 1.00E-08 1712.415389
2.5 0.391666667 271.61 693.4723404 1.00E-07 2772.004399
2.5 0.371 247.67 667.574124 1.00E-05 2668.481928
3.5 0.471666667 4.409 9.34770318 0 37.36540425
3.5 0.499 5.094 10.20841683 1.00E-10 40.80591931
3.5 0.557333333 28.99833333 52.03050239 1.00E-09 207.9805828
3.5 0.518 282.62 545.5984556 1.00E-08 2180.910803
3.5 0.514333333 463.9933333 902.1257291 1.00E-07 3606.050802
3.5 0.483333333 470.7433333 973.9517241 1.00E-05 3893.159548 
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

K176128.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.