Construction-Urea

1. Primers

All primers we used in this study were purchased from the Operon.

2. The Arg box

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

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

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

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)