Team:Tokyo Tech/Projects/Urea-cooler/construction
From 2011.igem.org
Construction-Urea
1. Primers
All primers we used in this study were purchased from the Operon.
2. The Arg box
![Argbox](https://static.igem.org/mediawiki/2011/0/07/For3_fig9.png)
The arg operator capable of binding the arginine repressor(Arg box) was amplified by PCR using the MG1655 as a template(1). The resulting fragment was ligated into Topo vector(2). To add restriction site, this was also amplified by PCR using a forward primer with EcoRI and XbaI sites, a reverse primer with SpeI and PstI sites(3).
3. The rocF
![rocF](https://static.igem.org/mediawiki/2011/e/e7/Urea1.png)
The rocF gene coding for arginage was amplified by PCR using a forward primer with an NcoI site, a reverse primer with SpeI and PstI sites, and the Bacillus subtilis as a template(4). The resulting fragment was cut with NcoI and PstI and ligated into pTrc99A backbone vector including trc promoter and RBS(5).
4. construction of Ptrc-RBS-rocF-Arg box
![Argbox plus](https://static.igem.org/mediawiki/2011/archive/5/54/20111028104906%21Urea2.png)
We cut Arg box(3) with XbaI and PstI and ligated into pTrc99A backbone vector including rocF(5) which were cut with SpeI and PstI(6). Then we amplified Ptrc-RBS-rocF-Arg box by PCR using a forward primer with EcoRI and XbaI sites, a reverse primer with SpeI and PstI sites(7). The resulting fragment was ligated into pSB3K3.(BBa_K649402)
5. construction of Ptrc-RBS-rocF
![Argbox minus](https://static.igem.org/mediawiki/2011/b/bf/Urea3.png)
We amplified Ptrc-RBS-rocF by PCR using a forward primer with EcoRI and XbaI sites, a reverse primer with SpeI and PstI sites, and using 5 as a template(8). The resulting fragment was ligated into pSB3K3.(BBa_K649301)