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Team:Tsinghua-A/Parts
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<P><Br></P> | <P><Br></P> | ||
| - | + | P ALIGN=LEFT STYLE="line-height: 0.58cm; widows: 2; orphans: 2"><FONT COLOR="#000000"><FONT FACE="Arial Unicode MS, sans-serif"><FONT SIZE=3 STYLE="font-size: 11pt"><SPAN LANG="en-US">Description: | |
| - | + | </SPAN></FONT></FONT></FONT> | |
| - | <P ALIGN=LEFT> | + | </P> |
| - | + | <P ALIGN=LEFT STYLE="line-height: 0.58cm; widows: 2; orphans: 2"><FONT COLOR="#000000"><FONT FACE="Arial Unicode MS, sans-serif"><FONT SIZE=3 STYLE="font-size: 11pt"><SPAN LANG="en-US">We | |
| - | + | assembled <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_E0840">BBa_E0840</A> | |
| - | + | under the pLas promoter <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_R0079">BBa_R0079</A> | |
| - | < | + | that was contained into K574009. We kept pSB1A2 as the scaffold |
| - | + | vector.<BR></SPAN></FONT></FONT></FONT><BR><BR> | |
| - | 1. | + | </P> |
| - | 2. | + | <P ALIGN=LEFT STYLE="line-height: 0.58cm; widows: 2; orphans: 2"><FONT COLOR="#000000"><FONT FACE="Arial Unicode MS, sans-serif"><FONT SIZE=3 STYLE="font-size: 11pt"><SPAN LANG="en-US">Methods:<BR>1. |
| - | 3. | + | Ligate <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_E0840">BBa_E0840</A> |
| - | 4. | + | downstream of <A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K574009">BBa_K574009</A>, |
| - | 5. | + | which acts as a reporter.<BR>2. Culture the positive colony at 37°C, |
| - | 6. | + | 220 rpm for 12 hours, as well as the DH5alpha.<BR>3. Dilute 1:25 the |
| - | < | + | former in 12 falcon tubes (4 ml cultures), each 3 tubes as a group. |
| - | </ | + | The later is also diluted in 3 tubes as the negative control.<BR>4. |
| - | + | After culturing for another 2 hours, as their OD600 reach 0.2, induce | |
| - | < | + | with 3OC12HSL 0, 1e-6 M, 1e-5 M and 1e-4 M respectively.<BR>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).<BR>6. Measure the fluorescence of | |
| - | < | + | the samples with the flow cytometer.</SPAN></FONT></FONT></FONT></P> |
| - | + | <P STYLE="line-height: 0.58cm; widows: 2; orphans: 2"><FONT FACE="Times New Roman, serif"><SPAN LANG="en-US"><FONT COLOR="#000000"><FONT FACE="Arial, Helvetica, sans-serif"><FONT SIZE=3 STYLE="font-size: 11pt"><SPAN STYLE="font-style: normal"><SPAN STYLE="font-weight: normal"><BR><BR><BR>Results:</SPAN></SPAN></FONT></FONT></FONT></SPAN></FONT></P> | |
| - | + | <P STYLE="line-height: 0.58cm; widows: 2; orphans: 2"><FONT FACE="Times New Roman, serif"><SPAN LANG="en-US"><FONT COLOR="#000000"><FONT FACE="Arial, Helvetica, sans-serif"><FONT SIZE=3 STYLE="font-size: 11pt"><SPAN STYLE="font-style: normal"><SPAN STYLE="font-weight: normal">From | |
| - | </SPAN></FONT></P> | + | 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^-6M, and to most groups, the intensity of GFP | ||
| + | reached its maxium after 4 hours.</SPAN></SPAN></FONT></FONT></FONT></SPAN></FONT></P> | ||
Revision as of 15:04, 5 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 |
Parts Data
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 12 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-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^-6M, and to most groups, the intensity of GFP reached its maxium after 4 hours.
