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Team:Tsinghua-A/Parts
From 2011.igem.org
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<h1>Descriptions</h1><hr width="70%" size=2 color=gray> | <h1>Descriptions</h1><hr width="70%" size=2 color=gray> | ||
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| - | < | + | <p>We assembled <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_E0840"><FONT COLOR="#002bb8">BBa_E0840</FONT></A> |
| + | under the pLas promoter | ||
| + | <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_R0079"><FONT COLOR="#002bb8">BBa_R0079</FONT></A> | ||
| + | that was contained into K574009. We kept pSB1A2 as the scaffold vector.</p> | ||
| - | < | + | <h1>Methods</h1><hr width="70%" size=2 color=gray> |
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| - | <p | + | <p>1. Ligate |
| + | <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_E0840"><FONT COLOR="#002bb8">BBa_E0840</FONT></A> | ||
| + | downstream of | ||
| + | <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K574009"><FONT COLOR="#002bb8">BBa_K574009</FONT></A> | ||
| + | , which acts as a reporter.</p> | ||
| + | <p>2. Culture the positive colony at 37°C, | ||
| + | 220 rpm for 12 hours, as well as the DH5alpha.</p> | ||
| - | < | + | <p>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.</p> | ||
| + | <p>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.</p> | ||
| + | |||
| + | <p>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).</p> | ||
| + | |||
| + | <P>6. Measure the fluorescence of the samples with the flow cytometer.</P> | ||
| + | |||
| + | <h1>Results</h1><hr width="70%" size=2 color=gray> | ||
| + | <p style="text-indent:0em"align="CENTER"><img src="https://static.igem.org/mediawiki/2011/c/ca/K574009_chart.png" NAME="K574009_chart" /></p> | ||
<P>From | <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 | ||
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Revision as of 07:55, 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.
