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Team:Tsinghua-A/Parts
From 2011.igem.org
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<h1 id="Dimensionless process">Results</h1><hr width="70%" size=2 color=gray> | <h1 id="Dimensionless process">Results</h1><hr width="70%" size=2 color=gray> | ||
| - | <P | + | <P>From |
| - | + | ||
the chart, we can see that cells were hardly induced in the control | the chart, we can see that cells were hardly induced in the control | ||
group, and with the concentration of inducer growing, the intensity | group, and with the concentration of inducer growing, the intensity | ||
of GFP increased by groups. The most efficient concentration of | of GFP increased by groups. The most efficient concentration of | ||
| - | inducer was around 10 | + | inducer was around 10<sup>-5</sup>M, and higher concentration may lead to the |
| - | reached its maxium after 4 hours. | + | expression of GFP decreasing. Additionally, to most groups, the intensity of GFP |
| + | reached its maxium after 4 hours.</P> | ||
Revision as of 07:44, 26 October 2011
Parts List
|
Name |
Type |
Description |
Length |
|---|---|---|---|
|
Composite |
pLux + TetR |
903 |
|
|
Composite |
TetR regulated by 3OC6HSL |
1890 |
|
|
Composite |
pluxR + CFP |
941 |
|
|
Device |
3OC6HSL -> TetR && reporter |
2839 |
|
|
Composite |
3OC12HSL regulated by TetR |
797 |
|
|
Composite |
3OC12HSL and YFP regulated by pBad |
1712 |
|
|
Device |
3OC12HSL regulated by pTet and pBad |
2517 |
|
|
Composite |
lasR + TT |
918 |
|
|
Composite |
a constitutive LasR producer |
982 |
|
|
Composite |
3OC12HSL -> PoPS Receiver |
1147 |
Descriptions
We assembled BBa_E0840 under the pLas promoter BBa_R0079 that was contained into K574009. We kept pSB1A2 as the scaffold vector.
Methods
1.
Ligate BBa_E0840 downstream
of BBa_K574009,
which acts as a reporter.
2. Culture the positive colony at 37°C,
220 rpm for 12 hours, as well as the DH5alpha.
3. Dilute 1:25 the
former in 15 falcon tubes (4 ml cultures), each 3 tubes as a group.
The later is also diluted in 3 tubes as the negative control.
4.
After culturing for another 2 hours, as their OD600 reach 0.2, induce
with 3OC12HSL 0, 1e-7 M, 1e-6 M, 1e-5 M and 1e-4 M respectively.
5. Take
samples after 1 hour, 4 hours, 8 hours and 24 hours, that is,
centrifugalizate 0.5 mL of each culture and suspend them by 1 mL of
PBS (phosphate-buffered saline).
6. Measure the fluorescence of
the samples with the flow cytometer.
Results
From the chart, we can see that cells were hardly induced in the control group, and with the concentration of inducer growing, the intensity of GFP increased by groups. The most efficient concentration of inducer was around 10-5M, and higher concentration may lead to the expression of GFP decreasing. Additionally, to most groups, the intensity of GFP reached its maxium after 4 hours.
