Part:BBa_K4677005
TorCAD promoter-LacZ
TorCAD promoter containing operon sequence is directly regulated by TorR protein in E.coli. TorCAD promoter can transcribe TMAO reductase genes including TorC, TorA and TorD. TorC codes for a membrane-bound c-type cytochrome with five heme-binding sites, TorA is the structural gene of TMAO reductase, and the product of TorD is a TorA specific chaperone. The reduction of TMAO by reductase allows anaerobic growth on nonfermentable sources, such as glycerol, mainly due to the TMAO reductase pathway. In the presence of TMAO, the sensor protein complex TorT-TorS bind with TMAO and transphosphorylate the response regulator TorR, activating torCAD promoter to transcribe TorC, TorA and TorD. The torR gene is located just upstream of the torCAD operon, with an opposite transcription direction. The torR-torCAD intergenic region is unusual in that it contains four direct repeats (the tor boxes) of a 10-nucleotide motif. Part or all of these motifs could be involved in the binding of TorR protein. The tor box1-box2 region constitutes a TorR high-affinity binding site, whereas box3 and box4 correspond to low-affinity binding sites. Phosphorylated as well as unphosphorylated TorR bind the box 1-box2 region, which seems to allow cooperative binding of phosphorylated TorR to box3 and box4. The cloned TorCAD promoter contains all of these four motifs to bind TorR, regulating the torCAD promoter. To quantify the activity of torCAD promoter, we cloned torCAD-LacZ to express β-galactosidase which catalyzes the subtrate X-gal, generating blue color product. With co-transformation of two plasmids pET28a-TorR-LacZ (containing torCAD) and pET22b-TorT-TorS, in the presence of TMAO, the activity of torCAD promoter was quantified.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
In order to construct the standard part pSB1C3-TorCAD-LacZ plasmid, TorCAD-LacZ sequence was detected to see whether there is EcoRI and PstI site. The detecting result was showed in Fig.1.
Fig.1 The map of TorCAD-LacZ sequence described by SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).
After detecting the restriction enzyme information of TorCAD-LacZ using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-TorCAD-LacZ with PCR method. Then it was identified as follows:
Fig.2 Identification of standard part pSB1C3-TorCAD-LacZ using digestion with EcoRI and PstI, and PCR method.
M: Marker; 1: Plasmid; 2: Digestion result; 3: PCR result.
Fig.3 The structure and function of genes involved in the TMAO reductase pathway.
Fig.4 The principle of that X-gal is catalyzed to produce blue product, detecting the expression of LacZ (β-galactosidase) regulated by TorCAD promoter.
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