Part:BBa_K5160111
pET28a - T7 promoter-6x His-brazzein-6x His-T7 terminator
Overview
This year, SZU-China embarked on an exciting project—developing an innovative system for producing sugar substitutes using tomatoes. By applying transgenic technology and the E8 specific promoter, we successfully achieved efficient production of Thaumatin in tomato fruits. Additionally, we introduced the SPS-NTPP vacuolar targeting peptide at the N-terminal of Thaumatin, allowing targeted storage of the sweet protein and increasing its concentration in tomatoes.
During our exploration of sweet proteins, we identified Brazzein as a notable candidate due to its impressive properties. However, because Brazzein currently lacks safety certification, it cannot yet be marketed or consumed. Despite this limitation, we proceeded with its characterization in the experimental phase and completed the relevant part forms, hoping that it will see widespread use in the future.
Brazzein, a sweet protein derived from the Pentadiplandra brazzeana Baillon plant found in the tropical African rainforest, is notable for its strong thermal stability and acid resistance. It binds to the T1R2 and T1R3 sweet taste receptors on the human tongue, creating a sweet sensation. Moreover, Brazzein is fully digestible, breaking down into common amino acids without producing significant calories, making it a highly promising sweetener.
Given its potential, we also characterized Brazzein in hopes that this novel sugar substitute will eventually gain global approval. In the early stages of the experiment, we explored its expression in the prokaryotic organism E. coli.
Learn more in (Engineering).
Team SZU-China 2024 has constructed a pET28a - T7 promoter-6x His-brazzein-6x His-T7 terminator vector to explore whether brazzein can be correctly folded and expressed in prokaryotes. This is the first step in our exploration, and we use it as a foundation to drive the optimization of subsequent genetic pathways and chassis selection. The following text provides a detailed description of the characterization of brazzein produced in E. coli this year. We hope that these characterizations can provide the iGEM community with more information about the heterologous expression of proteins in prokaryotes and offer inspiration and suggestions for future iGEM teams interested in this area.
Sequence and Features
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- 21COMPATIBLE WITH RFC[21]
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- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 270
Usage and Biology
In order to mass-produce our target protein Brazzein to meet market demand, we have set our sights on the commonly used model organism in prokaryotes - E. coli BL21(DE3). As a frequently used cellular factory in synthetic biology, E. coli BL21(DE3) has the characteristics of rapid growth and reproduction, clear genetic background, and low cost. Therefore, we have constructed a pET28a - T7 promoter-6x His-Brazzein-6x His-T7 terminator plasmid and integrated the target gene sequence into the E. coli genome to achieve heterologous expression of the sweet protein Brazzein in E. coli BE21(DE3).
T7 promoter
The T7 promoter is a strong promoter derived from the T7 phage that reacts specifically to T7 RNA polymerase and is a sequence that initiates the transcription of T7 phage genes. In the fields of 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 Brazzein, we can induce the expression of the target gene Brazzein in E. coli. For more detailed information about this part, 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 is usually composed of 6 to 10 consecutive histidine residues, and the most commonly used His tag is the 6x His tag, with a molecular weight of about 0.8 kDa. As a commonly used tag in scientific research, the molecular weight of the His protein is small 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 to detect the target protein and use six of them to improve the detection level. For more detailed information about this part, please refer to BBa_K157011.
Brazzein
Brazzein comes from the pulp of Pentadiplandra brazzeana Baillon (P. brazzeana), which grows in the tropical rainforests of Africa. Each fruit has a red fruit shell-like outer pericarp, under which there are three to five kidney-shaped seeds surrounded by a thick layer of soft red pulp, which contains Brazzein. Brazzein is the smallest sweet protein, with a peptide chain composed of 54 amino acids, and a molecular weight of only 6.5 kDa.
Brazzein triggers the sweet taste sensation by binding to the T1R2 and T1R3 sweet taste receptors on the human tongue. At the same time, the flavor characteristics of Brazzein are similar to sucrose, and its sweetness is 500-2000 times that of sucrose. In addition, Brazzein is a protein with excellent heat stability and acid resistance. It can maintain its protein properties after being incubated at 98°C for 2 hours, at 80°C for 4.5 hours, and within the pH range of 2.5-8 at 80°C for 4 hours. For more detailed information about this part, please refer to BBa_K5160004.
T7 terminator
The T7 terminator is a specific DNA sequence used in molecular biology to terminate the transcription process. The T7 terminator is a sequence-specific element in the T7 phage genome that determines the site where the transcription unit stops 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, thus 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 more detailed information about this part, please refer to BBa_M50060.
Structural Design
We have successfully constructed the gene sequence of T7 promoter-6x His-Brazzein-6x His-T7 terminator on the pET-28(+) plasmid and integrated the expression vector into the genome of E. coli using the expression vector. The whole gene synthesis technology transforms the aforementioned expression vector into E. coli BL21 (DE3), and single-clone bacteria are picked and expanded in a culture medium containing Kan (kanamycin).
Characterization
To verify whether E. coli BL21 (DE3) can correctly express the target gene Brazzein, we picked single-clone bacteria from a culture medium containing Kan (kanamycin), expanded them, and extracted bacterial proteins for Western Blot (WB) experiments to detect the sweet protein. The results (Fig 4.) showed that Brazzein was expressed in E. coli, with its low expression level, which may be due to the limitations of the prokaryotic expression system of E. coli and the formation of inclusion bodies. This experiment can prove that the sweet protein expressed by E. coli is not effective, therefore we need to optimize the selection of the chassis.
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 have constructed the gene sequence of T7 promoter-6x His-Brazzein-6x His-T7 terminator on the pET-28(+) plasmid and integrated the expression vector into the genome of Escherichia coli using the expression vector. Western Blot (WB) was used to verify the successful expression of the heterologous protein Brazzein, but the expression results were unsatisfactory. On one hand, since Brazzein is a plant-derived protein, it is difficult to correctly fold and express in large quantities in prokaryotic organisms. On the other hand, E. coli requires extraction and purification to obtain the target protein, which may lead to protein contamination and raise concerns about food safety. Therefore, this has inspired us to change the chassis organism and continue to optimize the genetic route. Subsequently, we attempted to express it in a plant chassis - tomatoes, and optimized the genetic route.
Reference
[1] Assadi-Porter FM, Maillet EL, Radek JT, Quijada J, Markley JL, Max M. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor. J Mol Biol. 2010 May 14;398(4):584-99. doi: 10.1016/j.jmb.2010.03.017. Epub 2010 Mar 17. PMID: 20302879; PMCID: PMC2879441.
[2] Markova EV, Leonova EI, Sopova JV. [Sweet protein brazzein as a promising sweetener]. Vopr Pitan. 2024;93(1):61-71. Russian. doi: 10.33029/0042-8833-2024-93-1-61-71. Epub 2024 Jan 19. PMID: 38555610.
[3] Saraiva A, Carrascosa C, Ramos F, Raheem D, Pedreiro S, Vega A, Raposo A. Brazzein and Monellin: Chemical Analysis, Food Industry Applications, Safety and Quality Control, Nutritional Profile and Health Impacts. Foods. 2023 May 10;12(10):1943. doi: 10.3390/foods12101943. PMID: 37238762; PMCID: PMC10217172.
[4] Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta. 2005 Jun;221(4):523-30. doi: 10.1007/s00425-004-1466-4. Epub 2005 Jan 29. PMID: 15682278.
[5] 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.
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