Part:BBa_K4677003

TorT-TorS

The TMAO reductase pathway is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. TorT associates as a coreceptor with the histidine kinase receptor TorS to form the sensor complex. TorT-TorS is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase. TMAO-stimulated TorS phosphorylates the response regulator TorR; phosphorylated TorR activates transcription from the torCAD operon to express TorCAD which are involved in the TMAO reductase pathway.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1361
  Illegal XhoI site found at 3615
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 42
  Illegal AgeI site found at 3366
  
• 1000
  COMPATIBLE WITH RFC[1000]

The TMAO reductase pathway involves some proteins in E.coli. To understand this pathway of TorCAD expression regulated by TorR depedently on TMAO, we drew a schematic diagram to show this process (Fig.1). In the presence of TMAO, TorT-TorS form an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase. TMAO-stimulated TorS phosphorylates the response regulator TorR; phosphorylated TorR activates transcription from the torCAD operon to express TorC, TorA and TorD in which TorA encodes a structural reductase, catalyzing TMAO reduction to allow anaerobic growth on nonfermentable sources.

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Fig.1 The TMAO reductase pathway to show the TorCAD expression regulated by TorR depedently on TMAO.

To deeply understand the stucture of TorT-TorS complex and TMAO binding site, Fig.2 was cited from refference [1].

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Fig.2 Structure of the TorT-TorS Complex and TMAO Binding Site. (a): Ribbon diagram of the complex viewed down dyad axis into the membrane. (b) : Ribbon diagrams of the TMAO binding sites (Asp42, Tyr44, Trp45, Tyr71, Trp140, and Tyr252) of TorT.

Since TorR-LacZ were constructed into pET-28a (kan+) plasmid, and co-transformation of TorR-LacZ with TorT-TorS are required for LacZ expression in cells, the TorT-TorS genes were designed to insert into pET-22b (Amp+) for different antibiotic screening. TorT gene contains 1029bp, and TorS gene contains 2715bp which are arranged in the same chromosome with an opposite transcriptional direction (Fig.3).

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Fig.3 The structure of TorT, TorS and TorR gene with TorCAD promoter, indicating the opposite arrangement of TorT and TorS in the same chromosome.

Considering that it is easy to purify the recombinant protein expressed by the promoter of pET-22b expression vector, TorT and TorS are designed to construct into pET-22b plasmid. To simplify the construction of the two genes, TorT and TorS are designed to recombine together using a RBS sequence followed by extra 6 bases, making them transcribed together (controlled by the same promoter), but translated separately (Fig.4).

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Fig.4 TorT and TorS genes were designed to construct into pET-22b vector, forming a fusion gene.

TorT and TorS were synthesized and flanked with BamH I and Hind III sites, then inserted into pET-22b vector, constructing pET-22b-TorT-TorS. For identification, TorT-TorS was amplified by PCR method and digestion the recombinant plasmid using BamH I and Hind III. The result was shown in Fig.5.

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Fig.5 Identification of pET-22b-TorT-TorS plasmid. M: Marker; 1: The plasmid of pET-22b-TorT-TorS; 2: The pET-22b-TorT-TorS plasmid digested by BamH I and Hind Ⅲ restriction endonuclease; 3: The TorT-TorS genes amplified with PCR method.

For testing whether the pET-22b-TorT-TorS express TorT and TorS proteins or not, it was transformed to BL21 strain, after culturing about 20h with IPTG induction, cells were collected by centrifugation. Soluble cytoplasmic proteins were extracted after lysis and purified using 6x His tag. Then SDS-PAGE electrophoresis was performed. The expected bands (TorT is about 40kD, and TorS is about 110kD) were observed, which was shown in Fig.6.

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Fig.6 The SDS-PAGE result shows expression and purification of TorT and TorS proteins. M: Marker; 1: All supernatant proteins containing TorT and TorS induced by IPTG; 2: Supernatant proteins not bound to magnetic beads in purification process; 3: Proteins in wash buffer; 4: Purified TorT (40kD) and TorS (110kD) proteins in elution buffer.

From the result above, we know that the recombinant plasmid pET-22b-TorT-TorS was constructed successfully. It can be used for detection TorCAD promoter activity regulated by TorR dependently on TMAO concentration. To contribute to the iGEM community in future, we built a standard part (BioBrick) in the follwing.

In order to construct the standard part pSB1C3-TorT-TorS plasmid, TorT-TorS sequence was tested to see if there is EcoRI and PstI site. The testing result was showed in Fig.7.

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Fig.7 The map of TorT-TorS sequence described by SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).

After testing the restriction enzyme information of TorT-TorS gene using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part (BioBrick) pSB1C3- TorT-TorS with PCR method. Then it was identified as follows (Fig.8):

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Fig.8 Identification of standard part pSB1C3-TorT-TorS using PCR method and digestion with EcoR I and Pst I. M: Marker; 1: Plasmid; 2: PCR result; 3: Digestion result.

Summary

This part (K4677003) was aimed to construct for co-transformation with pET-28a-TorR-LacZ plasmid, detecting 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 (Fig.9), which could be quantified. For more information, please refer to our another part K4677005 (Part:BBa K4677005 - parts.igem.org) or our result (Results | HSASNU - iGEM 2023).

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Fig.9 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).

Refferences

[1] Moore JO, Hendrickson WA. An asymmetry-to-symmetry switch in signal transmission by the histidine kinase receptor for TMAO. Structure. 2012; 20(4): 729-741.doi:10.1016/j.str.2012.02.021.
[2] Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013; 368(17):1575-1584.
[3] Senthong V, Wang Z, Fan Y, Wu Y, Hazen SL, Tang WH. Trimethylamine N-Oxide and Mortality Risk in Patients With Peripheral Artery Disease. J Am Heart Assoc 2016; 5(10).
[4] Senthong V, Wang Z, Li XS, Fan Y, Wu Y, Tang WH, et al. Intestinal Microbiota-Generated Metabolite Trimethylamine-N-Oxide and 5-Year Mortality Risk in Stable Coronary Artery Disease: The Contributory Role of Intestinal Microbiota in a COURAGE-Like Patient Cohort. J Am Heart Assoc 2016; 5(6).
[5] Zhu Y, Li Q, Jiang H. Gut microbiota in atherosclerosis: focus on trimethylamine N-oxide. APMIS. 2020;128(5):353-366. doi:10.1111/apm.13038.