Difference between revisions of "Part:BBa K176130"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K176130 short</partinfo>
 
<partinfo>BBa_K176130 short</partinfo>
Line 17: Line 16:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K176130 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K176130 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===
 +
'''K176130'''
 +
{| {{table}}
 +
| 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||0.140666667||0.832666667||5.91943128||0||23.66163521
 +
|-
 +
| 0.5||0.146666667||0.747666667||5.097727273||1.00E-10||20.3770527
 +
|-
 +
| 0.5||0.151||1.635||10.82781457||1.00E-09||43.28182664
 +
|-
 +
| 0.5||0.144666667||5.035333333||34.80645161||1.00E-08||139.1311972
 +
|-
 +
| 0.5||0.145||6.813666667||46.9908046||1.00E-07||187.8354903
 +
|-
 +
| 0.5||0.147||7.474||50.84353741||1.00E-05||203.2359492
 +
|-
 +
| 1.5||0.249666667||1.616333333||6.473965287||0||25.87826393
 +
|-
 +
| 1.5||0.262||1.293666667||4.937659033||1.00E-10||19.73721483
 +
|-
 +
| 1.5||0.266333333||1.912666667||7.181476846||1.00E-09||28.70638704
 +
|-
 +
| 1.5||0.256||9.243||36.10546875||1.00E-08||144.3237349
 +
|-
 +
| 1.5||0.248666667||26.90566667||108.1997319||1.00E-07||432.5048243
 +
|-
 +
| 1.5||0.243||52.47433333||215.9437586||1.00E-05||863.1880664
 +
|-
 +
| 2.5||0.407||3.353666667||8.23996724||0||32.93747148
 +
|-
 +
| 2.5||0.414||2.235666667||5.400161031||1.00E-10||21.58596567
 +
|-
 +
| 2.5||0.424||3.147333333||7.422955975||1.00E-09||29.67164718
 +
|-
 +
| 2.5||0.41||15.49433333||37.79105691||1.00E-08||151.0615058
 +
|-
 +
| 2.5||0.394333333||55.61766667||141.0422654||1.00E-07||563.7856874
 +
|-
 +
| 2.5||0.39||139.1066667||356.6837607||1.00E-05||1425.765522
 +
|-
 +
| 3.5||0.565666667||9.078||16.04832057||0||64.14966049
 +
|-
 +
| 3.5||0.577333333||3.753||6.500577367||1.00E-10||25.98463991
 +
|-
 +
| 3.5||0.607||5.145333333||8.476661175||1.00E-09||33.88360385
 +
|-
 +
| 3.5||0.579666667||26.34833333||45.45428407||1.00E-08||181.6935846
 +
|-
 +
| 3.5||0.557666667||98.57133333||176.7567244||1.00E-07||706.5464462
 +
|-
 +
| 3.5||0.558||267.6666667||479.6893668||1.00E-05||1917.453599
 +
|}
 +
For the details of the definition of '''ST.FLU''' and the unit '''ustc_st1''', please click [[here]].
 +
 +
===plot===
 +
 +
[[Image:K176130.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 08:31, 20 October 2009

TetR repressible AHL->GFP Receiver (Low LuxR): pCon 0.04->luxR+pLux/Tet->GFP

AHL -> GFP Receiver

TetR repressible

Low LuxR level

K176129 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

K176130

T(hour) OD600 FLU(arbitrary) FLU/OD(arbitrary) AHL ST.FLU/OD(ustc_st1)
0.5 0.140666667 0.832666667 5.91943128 0 23.66163521
0.5 0.146666667 0.747666667 5.097727273 1.00E-10 20.3770527
0.5 0.151 1.635 10.82781457 1.00E-09 43.28182664
0.5 0.144666667 5.035333333 34.80645161 1.00E-08 139.1311972
0.5 0.145 6.813666667 46.9908046 1.00E-07 187.8354903
0.5 0.147 7.474 50.84353741 1.00E-05 203.2359492
1.5 0.249666667 1.616333333 6.473965287 0 25.87826393
1.5 0.262 1.293666667 4.937659033 1.00E-10 19.73721483
1.5 0.266333333 1.912666667 7.181476846 1.00E-09 28.70638704
1.5 0.256 9.243 36.10546875 1.00E-08 144.3237349
1.5 0.248666667 26.90566667 108.1997319 1.00E-07 432.5048243
1.5 0.243 52.47433333 215.9437586 1.00E-05 863.1880664
2.5 0.407 3.353666667 8.23996724 0 32.93747148
2.5 0.414 2.235666667 5.400161031 1.00E-10 21.58596567
2.5 0.424 3.147333333 7.422955975 1.00E-09 29.67164718
2.5 0.41 15.49433333 37.79105691 1.00E-08 151.0615058
2.5 0.394333333 55.61766667 141.0422654 1.00E-07 563.7856874
2.5 0.39 139.1066667 356.6837607 1.00E-05 1425.765522
3.5 0.565666667 9.078 16.04832057 0 64.14966049
3.5 0.577333333 3.753 6.500577367 1.00E-10 25.98463991
3.5 0.607 5.145333333 8.476661175 1.00E-09 33.88360385
3.5 0.579666667 26.34833333 45.45428407 1.00E-08 181.6935846
3.5 0.557666667 98.57133333 176.7567244 1.00E-07 706.5464462
3.5 0.558 267.6666667 479.6893668 1.00E-05 1917.453599 
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

K176130.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.