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In this competition season, we constructed some parts which are involved in our project. Here, we use the new part BBa_K4677003 (Part: BBa K4677003 - parts.igem.org) as an example to demonstrate our engineering success, following the engineering design cycle to show our effort. |
Background
The TMAO reductase pathway involves some proteins in E.coli. To understand this pathway of TorCAD expression regulated by TorR dependently 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 cooperatives 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.
Fig.1 The TMAO reductase pathway to show the TorCAD expression regulated by TorR dependently on TMAO.
To deeply understand the structure of TorT-TorS complex and TMAO binding site, Fig.2 was cited from reference [1].
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.
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Engineering problem
To establish the filter paper sensor to detect TMAO, we need to construct some recombinant plasmids containing TorT and TorS genes. As we know from Fig.1, TorT-TorS form a complex heterotetramer, in order to reduce the number of constructed plasmids, we planned that TorT and TorS genes are recombinant together for construction. But how to design it?
Engineering process
| Design StageSince 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 1029 bp, and TorS gene contains 2715 bp which are arranged in the same chromosome with an opposite transcriptional direction (Fig.3). |
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.
To simplify the construction of the two genes, TorT and TorS are designed to recombine together to form a fusion gene transcribed by a same promoter of the vector. After transcription and translation, the fusion protein would be digested to separate each other. With this consideration, the fusion gene TorT and TorS are designed to insert into pET-22b vector (Fig.4).
Fig.4 TorT and TorS genes were designed to construct into pET-22b vector, forming a fusion gene.
| Build StageTorT and TorS fusion gene was synthesized and flanked with BamH I and Hind III sites. After PCR amplification and restriction endonuclease digestion, TorT and TorS fusion gene was successfully 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. |
Fig.5 pET-22b-TorT-TorS was identified using PCR method and restriction endonuclease digestion by BamH I and Hind III.
M: Marker; 1: Plasmid; 2: PCR result; 3: Digestion result.
| Test StageFor testing whether the pET-22b-TorT-TorS express TorT and TorS proteins or not, it was transformed to BL21 strain, after culturing about 20 h with IPTG induction using fresh LB medium added with Ampicillin, Cells were collected by centrifugation. Soluble cytoplasmic proteins were extracted after lysis and purified for identification using 6x His tag. Then SDS-PAGE electrophoresis was performed. However, the expected bands (TorT is about 40 kDa and TorS is about 110 kDa) were not observed, whereas there was a overexpressed protein band which molecule size is about 150 kDa (Fig.6). |
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 overexpressed protein（about 150 kDa）in elution buffer.
| Learn StageFrom the SDS-PAGE result, we figured out that the pET-22b-TorT-TorS could express TorT-TorS fusion protein. However it could not be digested to separate in cells, indicating a failed construction. So, we need to reconstruct pET-22b-TorT-TorS plasmid. |
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| Re-DesignTo get the separate TorT and TorS proteins, with more consideration, TorT and TorS genes are recombined using a RBS sequence followed by extra 6 bases, making them transcribed together (controlled by the same promoter), but translated separately (Fig.7). |
Fig.7 TorT and TorS genes are re-designed to combine together using RBS sequence followed by extra 6 bases, making them translated separately.
| Re-BuildTorT and TorS were designed to combine together using RBS (B0034) followed by extra 6 bases, synthesized and also flanked with BamH I and Hind III sites. Then they were inserted into pET-22b vector, constructing pET-22b-TorT-TorS again. The identification was performed by PCR and digestion with BamH I and Hind III. The result was shown in Fig.8. |
Fig.8 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.
| Re-TestTo test the new plasmid whether express the TorT and TorS proteins separately, same procedure was performed. Then SDS-PAGE electrophoresis was also conducted. As expected this time, two overexpressed bands (TorT 40 kDa and TorS 110 kDa) were observed in Fig.9. |
Fig.9 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 (40 kDa) and TorS (110 kDa) proteins in elution buffer.
| Re-LearnFrom the result of re-test, we know that the new recombinant plasmid pET-22b-TorT-TorS was constructed successfully. So, 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 in the following. |
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Construction of the standard part pSB1C3-TorT-TorS (K4677003)
In order to construct the standard part pSB1C3-TorT-TorS plasmid, TorT-TorS sequence was tested to see if there are EcoR I and Pst I sites. The testing result was shown in Fig.10.
Fig.10 The map of TorT-TorS sequence described by SnapGene Viewer, showing the restriction enzyme information (no EcoR I and Pst I 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.11):
Fig.11 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.
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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.12), 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).
Fig.12 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).
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References
[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