Part:BBa_K5160112
pET28a - T7 promoter-6x His-thaumatin-6x His-T7 terminator
Overview
This year, SZU-China has done something interesting - innovatively creating a tomato-based sugar substitute production system. By using genetically modified technology and under the induction of the E8 promoter, we have achieved efficient production of Thaumatin in tomato fruits. Simultaneously, by introducing the SPS-NTPP vacuolar targeting peptide at the N-terminus of Thaumatin, we have achieved targeted storage of the sweet protein, thereby increasing its content in tomatoes. Thaumatin, a sweet protein derived from the aril of the African rainforest plant Thaumatococcus danielli, binds to sweet taste receptors on the human tongue to produce a sweet sensation. It can be completely digested and broken down into common amino acids by the human body, generating almost no calories. However, due to its African plant origin, the current production of Thaumatin cannot meet the daily demands of people. Therefore, we have created this novel tomato system. However, in the early stages of the experiment, we conducted preliminary attempts at the prokaryotic organism Escherichia coli. For more detailed information, please refer to our Engineering.
The SZU-China 2024 team constructed the pET28a - T7 promoter-6x His-thaumatin-6x His-T7 terminator plasmid to explore whether Thaumatin could correctly fold and express in prokaryotic organisms. This was the first step in our exploration, and we used it as a foundation to drive subsequent optimizations in genetic pathways and host selection. The latter part of this document provides a detailed description of our characterization of Thaumatin production in Escherichia coli this year. We hope that these characterizations can provide the iGEM community with more information about heterologous protein expression in prokaryotic organisms, and offer inspiration and suggestions to future iGEM teams interested in this area.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 185
Illegal NotI site found at 222
Illegal NotI site found at 324 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 218
Illegal NgoMIV site found at 384 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 813
Usage and Biology
To achieve mass production of our target protein, Thaumatin, and thereby meet market demands, we focused on Escherichia coli BL21(DE3), a commonly used model organism in prokaryotes. As a cell factory frequently employed in synthetic biology, E. coli BL21(DE3) offers rapid growth and reproduction, a clear genetic background, and low costs. Therefore, we constructed the pET28a - T7 promoter-6x His-thaumatin-6x His-T7 terminator plasmid and integrated the target gene sequence into the E. coli genome to enable heterologous expression of the sweet protein Thaumatin in E. coli BL21(DE3).
T7 Promoter
The T7 promoter is a strong promoter derived from the T7 bacteriophage. It specifically reacts with T7 RNA polymerase and initiates the transcription of T7 bacteriophage genes. In molecular biology and biotechnology, the T7 promoter is often used to construct gene expression vectors. By regulating the expression or activity of T7 RNA polymerase, it controls the expression level of the target gene. In our design, by introducing the T7 promoter upstream of Thaumatin, we can induce the expression of the target gene Thaumatin in E. coli. For detailed information on this component, please refer to BBa_M50060.
6x His
The His protein tag, also known as the polyhistidine tag, is one of the most widely used affinity purification tags in recombinant protein purification. The His tag typically consists of 6 to 10 consecutive histidine residues, with the 6x His tag being the most commonly used, with a molecular weight of approximately 0.8 kDa. As a commonly used tag in research, the His protein has a small molecular weight and does not affect the structure and function of the recombinant protein. Therefore, it is often used for the purification and detection of recombinant proteins. In our project design, we use the His tag for the detection of the target protein and employ six His tags to enhance detection levels. For detailed information on this component, please refer to BBa_K157011.
Thaumatin
Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). There are two types of thaumatin in Thaumatococcus: thaumatin I and thaumatin II. Among them, thaumatin II has a higher sweetness, which is 3000 times that of sucrose. It is composed of 207 amino acids, with the amino acids folded into eight disulfide bonds. Therefore, the protein structure of thaumatin is stable, exhibiting thermal and acid stability.
Thaumatin belongs to the family of five pathogenesis-related proteins (PR5), characterized by its significant antifungal and plant pathogen activity. In Thaumatococcus, the main function of Thaumatin is to protect seeds and fruits against abiotic stresses. After discovering the sweet characteristics of Thaumatin, people have developed it for use in various foods. For detailed information on this component, please refer to BBa_K5160003.
T7 Terminator
The T7 terminator is a specific DNA sequence used in molecular biology to terminate the transcription process. It is a sequence-specific element in the T7 bacteriophage genome that determines the site where the transcription unit terminates transcription and initiates the process of separating the newly synthesized RNA from the transcription machinery. When using T7 RNA polymerase for gene expression, the T7 terminator plays a key role in ensuring that the transcription process stops accurately at a specific location, thereby avoiding interference with downstream genes. In prokaryotic expression systems such as E. coli, many expression vectors contain T7 promoter and T7 terminator sequences. This design allows researchers to precisely control the expression level of the target gene by regulating the expression of T7 RNA polymerase. In our design, we use the T7 terminator to control the expression of the target gene. For detailed information on this component, please refer to BBa_M50060.
Structural Design
We successfully constructed the gene sequence of T7 promoter-6x His-thaumatin-6x His-T7 terminator on the pET-28(+) plasmid and integrated it into the genome of Escherichia coli using an expression vector. Through whole-gene synthesis technology, the aforementioned expression vector was transformed into E. coli BL21 (DE3), and a monoclonal bacterium was selected and propagated in a medium containing Kan (kanamycin).
Characterization
To verify whether the target gene Thaumatin could be correctly expressed in E. coli BL21 (DE3), we picked a monoclonal bacterium from the medium containing kanamycin (Kan) for propagation and subsequently extracted bacterial proteins for Western blot (WB) analysis to detect the sweet protein. The results (Fig 3.) indicated that Thaumatin was expressed in E. coli, but the expression level was low. This may be attributed to the limitations of the prokaryotic expression system of E. coli and the formation of inclusion bodies. This experiment demonstrates that the sweet protein expressed in E. coli is not effective, necessitating the optimization of the host cell selection.
M1/M2: marker，PC1: BSA (1 μg)，PC2: BSA (2 μg)，NC: Cell lysate without induction，Lane 1: Cell lysate with induction for 16 h at 15 ℃，Lane 2: Cell lysate with induction for 4 h at 37 ℃ Lane NC1: Supernatant of cell lysate without induction，Lane 3: Supernatant of cell lysate with induction for 16 h at 15 ℃，Lane 4: Supernatant of cell lysate with induction for 4 h at 37 ℃，Lane NC2: Pellet of cell lysate without induction，Lane 5: Pellet of cell lysate with induction for 16 h at 15 ℃，Lane 6: Pellet of cell lysate with induction for 4 h at 37 ℃
Conclusion
We constructed the gene sequence of T7 promoter-6x His-thaumatin-6x His-T7 terminator on the pET-28(+) plasmid and integrated it into the genome of Escherichia coli using an expression vector. Through Western blot (WB) analysis, we verified the successful expression of the heterologous protein Thaumatin, but the expression results were not ideal and could not meet market demands. On the one hand, as Thaumatin is a plant-derived protein containing eight disulfide bonds, it is difficult to correctly fold and express in large quantities in prokaryotic organisms. On the other hand, E. coli requires extraction and purification processes to obtain the target protein, which may lead to protein contamination and compromise food safety. Therefore, this inspired us to switch to a different host organism and continuously optimize the gene route. Subsequently, we attempted expression in a plant host, tomato, and optimized the gene route by using the tomato fruit-specific ripening promoter E8 and the vacuolar signal peptide SPS-NTPP to increase the content of Thaumatin in tomato fruits.
Reference
[1] Transgenic Plants as Producers of Supersweet Protein Thaumatin II, Living reference work entry, 19 August 2016.
[2] de Jesús-Pires C, Ferreira-Neto JRC, Pacifico Bezerra-Neto J, Kido EA, de Oliveira Silva RL, Pandolfi V, Wanderley-Nogueira AC, Binneck E, da Costa AF, Pio-Ribeiro G, Pereira-Andrade G, Sittolin IM, Freire-Filho F, Benko-Iseppon AM. Plant Thaumatin-like Proteins: Function, Evolution and Biotechnological Applications. Curr Protein Pept Sci. 2020;21(1):36-51. doi: 10.2174/1389203720666190318164905. PMID: 30887921.
[3] Ohta K, Masuda T, Tani F, Kitabatake N. The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin. Biochem Biophys Res Commun. 2011 Mar 18;406(3):435-8. doi: 10.1016/j.bbrc.2011.02.063. Epub 2011 Feb 15. PMID: 21329673.
[4] Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113-30. doi: 10.1016/0022-2836(86)90385-2. PMID: 3537305.
[5] Hirai T, Kim YW, Kato K, Hiwasa-Tanase K, Ezura H. Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res. 2011 Dec;20(6):1285-92. doi: 10.1007/s11248-011-9495-9. Epub 2011 Feb 27. PMID: 21359850.
[6] Differential subcellular targeting of recombinant human a1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants,Plant Science,Volume 196, November 2012.
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