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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

In the presence of TMAO, the sensor protein complex TorT-TorS bind with TMAO and phosphorylate the response regulator TorR, binding with torCAD promoter to activate TorC, TorA and TorD transcription. To understand the mechanism of TorCAD expression regulated by TorR dependently on TMAO, we drew a schematic diagram to show this process (Fig.1).



Fig.1 The mechanism of TorCAD expression regulated by TorR dependently on TMAO. In the presence of TMAO, the sensor protein complex TorT-TorS bind with TMAO and phosphorylate the response regulator TorR which binds to TorCAD operon, activating torCAD promoter to transcribe TorC, TorA and TorD.

The TMAO reductase pathway involves many proteins of which genes are arranged complicatedly with an opposite transcription direction. To simplify the structure of these genes, we drew another schematic diagram to show the structure and functions of these genes (Fig.2).



Fig.2 A schematic diagram to show the structure and functions of genes involved in the TMAO reductase pathway.

From Fig.2, we can see that 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 in K4677005 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 substrate 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.

In order to construct the standard part (BioBrick) pSB1C3-TorCAD-LacZ plasmid, considering the enzyme sites in the prefix and sufix of the BioBrick, TorCAD-LacZ sequence was detected to see whether there is EcoR I and Pst I site. The detecting result was shown in Fig.3.



Fig.3 The map of TorCAD-LacZ sequence described by SnapGene Viewer, showing the restriction enzyme information (no EcoR I and Pst I sites).

After detecting the restriction enzyme information of TorCAD-LacZ using SnapGene software, it was amplified using PCR method and inserted into the pSB1C3 plasmid at EcoR I and Pst I sites, constructing the standard part pSB1C3-TorCAD-LacZ. Then it was identified using PCR and restriction endonucleases, the result was shown as follows (Fig.4):

Fig.4 Identification of standard part pSB1C3-TorCAD-LacZ using PCR method and restriction endonucleases digestion with EcoR I and Pst I. M: Marker; 1: Plasmid; 2: PCR result; 3 Digestion result.

The constructed part K4677005 is a composite part comprises 3 subparts K1086000 (TorCAD promoter), B0034 (RBS) and I732005 (LacZ coding sequence). It was aimed to be constructed to detect the activity of TorCAD promoter regulated by TorR in the presence of TMAO. LacZ serves as a reporter gene which encodes β-galactosidase to catalyze the substrate X-gal, generating blue product. The principle is shown in Fig.5.



Fig.5 The principle of that X-gal is catalyzed to produce blue product, detecting the expression of LacZ (β-galactosidase) regulated by TorCAD promoter.

For measure the activity of TorCAD promoter, we also cloned TorT-TorS recombinant plasmid (refer to part K4677003) which is required for TorR phosphorylaion (the active form). So, both TorR-torCAD promoter-LacZ (pET-28a-TorR-LacZ) and TorT-TorS genes (pET-22b-TorT-TorS) were co-transformed into BL21DlacZ strain (with LacZ deletion), In the presence of TMAO (100 µM) and X-gal (40 µg/mL), we observed the blue clones on the agar plate (Fig.6), indicating that both two constructed recombinant plasmids worked well.



Fig.6 The blue clones were observed on the agar plate when TorR-torCAD promoter-LacZ and TorT-TorS genes were co-transformed into BL21DLacZ in the presence of TMAO and X-gal. A: Negative control without TMAO; B: experiment group with TMAO (100 µM) and X-gal (40 µg/mL).

To detect the sensitive of TorCAD promoter to TMAO concentration, we quantified the blue product catalyzed by β-galactosidase. After co-transformation of pET-28a-TorR-lacZ and pET-22b-TorT-TorS plasmids into BL21DLacZ strain, it was cultured about 20 h with the induction of IPTG , then centrifuged and lysed with lysis buffer. The supernatant (cell free system) was collected after centrifugation again. Using the supernatant, we quantified the product catalyzed by reporter protein (β-galactosidase) in the presence of TMAO and X-gal to detect the sensitivity of TorCAD promoter to TMAO concentration. The results were shown and quantified in the followings(Fig.7), which indicated that TorCAD promoter is sensitive to TMAO concentration above 40 µM. All X-gal with different concentration worked well.



Fig.7 Detection and quantification of TorCAD promoter sensitivity to TMAO concentration in the presence of TMAO and X-gal using cell free system. (a) Concentration of X-gal: 1. 12 µg/mL; 2. 16 µg/mL; 3. 24 µg/mL; 4. 30 µg/mL; 5. 40 µg/mL; 6. 60 µg/mL; 7. 120 µg/mL; 8. 240 µg/mL. (b) Detectable concentration of TMAO: 1. 20 µM; 2. 40 µM; 3. 60 µM; 4. 80 µM; 5. 100 µM; 6. 200 µM; 7. 400 µM; 8. 800 µM. (c) Quantification analysis of X-gal. (d) Quantification analysis of TMAO.

Conclusion

From the results shown previously indicated that the constructed part K4677005 works well. It can be used for detection the TorCAD promoter’s activity in the presence of TMAO and X-gal.
(1) With the presence of TMAO (100 µM) and X-gal (40 µg/mL), some blue clones were observed on the agar plate.
(2) TorCAD promoter is sensitive to TMAO concentration above 40 µM. All X-gal with different concentration (from 12 µg/mL to 240 µg/mL) worked well.

Refferences

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