Part:BBa_K5160114
pTRV2-35S promoter-brazzein-3xflag-NOS terminator
Description
Brazzein, derived from Pentadiplandra brazzeanaBaillon (P. brazzeana) crops in tropical African rainforests, is a highly heat-stable as well as acid-tolerant sweet-tasting protein. It binds to the T1R2 and T1R3 sweet taste receptors on the human tongue to trigger sweet taste sensations. In addition, it can be fully digested by the body into common amino acids and absorbed, a process that generates little to no heat. Brazzein has not been approved, but because it is a small enough protein and is structurally stable enough to be developed by a number of biotechnology companies, it is clear that brazzein has great potential for application and may become a future leader in the field of sugar substitutes.
For more detailed information, please see our Engineering section.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 974
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 974
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1368
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 974
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 974
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 66
Illegal BsaI site found at 1092
Illegal BsaI.rc site found at 1371
The SZU-China 2024 team used this construct to validate the possibility of correct brazzein expression and folding in tomato. This was done to ensure that subsequent optimization of the pathway was in the right direction. With the development of biotechnology, brazzein has been attempted to be produced in biofermentation. However, fermentation production requires a protein purification process, which is complex and increases insecurity; even though some studies have suggested that this can be changed to some extent by yeast modification, strain modification requires long-term screening and evolution to achieve stability, a process that costs untold amounts of time and money. Therefore, our project developed a new production method. We have integrated brazzein production and storage based on tomato fruits. At the same time, we only need to get brazzein through a simple juicing process, which is convenient and safe.
Usage and biology
In order to verify the feasibility of the tomato sump, we need a fast test method. Therefore, we would like to accomplish this by transient expression. Many studies have shown that transient infestation can be achieved either by Agrobacterium immersion or by leaf injection of viral vectors.
Considering that our target was tomato fruits, it was clearly not feasible to achieve transient infestation by immersing the fruits in Agrobacterium. Therefore, we used TRV viral vector-mediated transient expression method. Plant viral expression vectors have been widely used for the regulation of plant gene expression and functional analysis. TRV as a double-stranded RNA virus, TRV1 contains the genetic information of the virus, and TRV2 contains the gene encoding the viral capsid protein, and the MCS sequence exists on TRV2 for cloning. Under the action of reverse transcriptase, the genome is reverse-transcribed into cDNA in vitro, and the cDNA is specifically modified and cloned to turn it into a vector that is not virulent but has both the ability to self-replicate and express exogenous genes. Subsequently, we combined TRV2 with sweet protein and injected it into tomato together with TRV1. When the virus replicates autonomously in tomato cells, it can utilize the material in tomato cells to express the sweet protein.
At the same time, we inhibited the RNA interference system of tomato plants themselves by introducing silencing repressor P19 to ensure that the viral vectors could remain in tomato for a period of time. In addition to this, in order to monitor the effectiveness of the viral vector, we introduced mCherry red fluorescent protein partial sequence as a control.
CaMV 35S promoter
CaMV 35S promoter refers to the 35S promoter from cauliflower mosaic virus (CaMV). This promoter directs 35S RNA synthesis during plant infection by CaMV and enables efficient expression in the tissues of many dicotyledonous plants. The CaMV 35S promoter acts as a constitutive promoter and initiates gene expression in all tissues. It is persistent, with relatively constant RNA and protein expression, but is not spatiotemporally specific. For more information about this part see BBa_K788000.
Brazzein
Brazzein is derived from the pulp of Pentadiplandra brazzeana Baillon (P. brazzeana), which grows in African rainforests. Each fruit has a red, husk-like exocarp with three to five kidney-shaped seeds surrounded by a thick layer of soft, red pulp, which contains brazzein, the smallest sweet-tasting protein, with a peptide chain consisting of 54 amino acids and a molecular weight of only 6.5 Kda. Brazzein is present in mature fruits in amounts ranging from 0.05 to 0.2% by weight. There are three forms of Brazzein, Brazzein I, Brazzein II and Brazzein III. Brazzein I accounts for 80% of the total Brazzein and contains pyroglutamic acid at the N-terminal end; Brazzein II contains glutamine at the N-terminal end, which is then converted to pyroglutamine. Type III Brazzein has a loss of N-terminal glutamine (or pyroglutamic acid).
Brazzein triggers the sweet taste sensation by binding to T1R2 and T1R3 sweet taste receptors on the human tongue. Brazzein also has a flavor profile similar to that of sucrose and is 500-2000 times sweeter than sucrose. The reason for this great sweetness is that brazzein binds to the sweetness receptor at multiple sites, and the main sites of interaction between brazzein and the sweetness receptor are Loop43 (site 1) and the N-terminal and C-terminal regions (site 2), and the nearby Loop33 and Loop9-19 (site 3) contribute significantly to the sweetness of brazzein.
Besides, Brazzein is a protein with excellent thermal stability and acid resistance. Due to its simple structure and four stable disulfide bonds, Brazzein exhibits high tolerance to high temperatures and extreme pH levels. It can maintain its protein properties after incubation at 98°C for 2 hours, at 80°C for 4.5 hours, and within a pH range of 2.5-8 at 80°C for 4 hours. Meanwhile, it also demonstrates outstanding thermal stability across a wide range of pH values. Furthermore, it is non-toxic and non-allergenic, and has been consumed by indigenous people in Africa for thousands of years.
In conclusion, Brazzein is an excellent sugar substitute.For more information about this part, please see BBa_K5160004.
3xflag tag
Flag tags utilize a short hydrophilic octameric acid peptide (DYKDDDDK) fused to the N-terminus or C-terminus of the target protein and usually do not interact with the target protein, so it does not affect the function of the target protein and is often used to detect and characterize overexpressed proteins. In our project, we use a 3x flag tag to improve detection. For more information about this part, please see BBa_K5160010.
NOS terminator
A terminator on a plant expression vector that terminates the transcription of a gene.For more information about this part, please see BBa_P10401.
Structural design
We utilized the TRV2-35S promoter-brazzein plasmid constructed from the virus TRV (Tobacco rattle virus) and infested tomato plants. We first transfected the plasmid into Agrobacterium GV3101 for amplification. To verify the success of the transformation, we performed pcr using specific primers and demonstrated the successful entry of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis.
Characterize
Agarose gel electrophoresis
The colonies were PCR amplified by specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the result graph, we can see that Brazzein (176bp) has a clear band near 100bp (Fig 6), thus verifying that our transformed Agrobacterium has already carried the target sequence and has the ability to express RNA, which plays a very important role in our further experiments.
RNA expression test
After ensuring that the plasmid was successfully transferred into Agrobacterium tumefaciens GV3101, we amplified Agrobacterium tumefaciens into culture and assembled the virus with MMA induction. Next, we injected the virus into the leaves of four-leaf-old micro-TOM. After the plants grew up, the leaves were collected for RNA extraction and RT-PCR experiments were performed to verify the transcription of the brazzein gene in the leaves. According to the results, it can be seen that the RNA band of brazzein (176 bp) has a band near 100 bp, which indicates that brazzein has started to be expressed in tomato plants, and we can proceed to the next step of verification (Fig 7).
Protein expression
We collected leaves and fruits of tomato plants infested with TRV virus and extracted proteins for WB assay. According to the results, it is known that the leaves, flowers and fruits of tomato were able to express brazzein (Fig 8).
Conclusion
In the tomato expression system, brazzein was able to be transcribed, translated, and folded normally, which indicates that plant chassis do have an advantage over microorganisms in expressing the protein. However, from the practical application point of view, there are still drawbacks of viral transient infiltration. Even though transient expression of viral vectors has been utilized in some studies to produce multiple recombinant proteins, the production method still requires purification, or else consumer concerns cannot be eliminated; On the other hand, transient expression does not allow for stable inheritance, which is far from being achieved if long-term production is desired. Therefore, we decided to switch the method and utilize the transgenic method to stably express the sweet protein. For more information about this part, please see BBa_K5160118 and BBa_K5160120.
Application Prospects
Considering that Brazzein does not currently have FDA safety approval to be offered to the general public as a food product. Therefore, we have only considered the possible future applications of Brazzein. We hope that this information will provide inspiration and help interested iGEM teams to promote the formation of their projects.
First, given the favorable nature of Brazzein, we hope that safety approvals such as nutritional evaluation and pathologic evaluation of Brazzein can be accelerated, thus promoting faster market entry of Brazzein. Secondly, we expect Brazzein to find a wide range of innovative applications in the food industry.
In our project, we provide a program for the innovative application of Brazzein. We propose a tomato sugar substitute production system that realizes mass production in tomatoes and solves the problem of low yield of Brazzein due to its plant origin. We believe that Brazzein has a bright future as a novel sweetener and can create a richer world of sweetness for the benefit of more people.
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