Part:BBa_K5160113
35S promoter_thaumatin_3xflag_NOS terminator
Description
In order to provide people with a safe sweetener, our project proposes a method for the efficient production of thaumatin, which we have decided to produce efficiently in tomato fruits. Thaumatin binds to receptors on the human tongue to induce sweetness, and in the process generates almost no heat. At the same time, it can be completely digested by the body and broken down into common amino acids for absorption. However, thaumatin's structure contains eight disulfide bonds. Through initial attempts using the TRV viral vector in tomatoes, we determined that replacing it into tomatoes to produce thaumatin was the better option. For more detailed information, please see our ENGINEERING!
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 950
Illegal NotI site found at 987
Illegal NotI site found at 1089 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1911
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 983
Illegal NgoMIV site found at 1149 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 66
Illegal BsaI site found at 1635
Illegal BsaI.rc site found at 1914
The SZU-China 2024 team used this construct to validate the possibility of proper thaumatin expression and folding in tomato. This was done to ensure that subsequent pathway optimization was in the right direction. With the development of biotechnology, thaumatin 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 a certain 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 thaumatin production and storage based on tomato fruits. At the same time, we only need to get thaumatin through a simple juicing process, which is convenient and safe.
Usage and biology
In order to verify the feasibility of tomato chassis, we need a fast inspection method. Therefore, we hope to achieve this goal by means of transient expression. Many studies show transient infection can be achieved through Agrobacterium immerse or by leaf injection using a viral vector.
Considering that our chasis is tomato fruit, it is obviously not feasible to immerse the fruit in Agrobacterium to achieve instantaneous infection. So, we used TRV viral vector-mediated transient expression method. Plant viral expression vectors have been widely used for expression regulation and functional analysis of plant genes. TRV as a double-stranded RNA virus, TRV1 contains the genetic information of the virus and TRV2 contains the gene coding for the viral capsid protein, and the MCS sequence is present 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 express the sweet protein using the material in tomato cells.
At the same time, we made sure that the viral vector could remain in tomato for a period of time by introducing silencing repressor P19 to inhibit the RNA interference system of the tomato plant itself. In addition to this, we introduced mCherry red fluorescent protein partial sequence as a control in order to monitor the effectiveness of the viral vector.
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.
Thaumatin
Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). Bamboo yam contains two types of thaumatin: thaumatin I and thaumatin II. One of them, thaumatin II, has a higher sweetness, which is 3,000 times that of sucrose. It consists of 207 amino acids, and the amino acids are coiled and folded to form eight disulfide bonds. As a result, thaumatin has a stable protein structure and is heat and acid stable.
Thaumatin belongs to the family of five pathogenesis-related proteins (PR5), which are characterized by significant antifungal as well as phytopathogenic activity. Thus, in bamboo yam, the main function of thaumatin is to protect seeds and fruits against abiotic stresses. The discovery of the sweet flavor characteristics of thaumatin has led to its development for use in a variety of food applications. For more information about this part, please see BBa_K5160003.
3x Flag 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-Thaumatin 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 proved the success of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis.
Characterize
Agarose gel electrophoresis
The colonies were amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the band of Thaumatin (719bp) has a band appearing near 700bp. (Fig 4) This can be verified that our transformed Agrobacterium has carried on the target sequence. This is an important milestone and lays the foundation for further experiments.
RNA expression test
After ensuring that the plasmid was successfully transferred into Agrobacterium GV3101, we amplified Agrobacterium into culture and assembled the virus by inducing it with MMA. 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 thaumatin gene in the leaves. According to the results, it can be seen that the RNA band of thaumatin (719 bp) has a band near 700 bp, which indicates that thaumatin has begun to be expressed in tomato plants, and we can proceed to the next step of verification. (Fig 5)
Protein expression
We collected leaves and fruits of TRV virus-infected tomato plants and extracted proteins for WB assay. According to the results, it was clear that tomato was able to express thaumatin (Fig 6). Based on the immunoenzymatic ELISA for thaumatin, we concluded that the average level of transient expression of thaumatin was 11.2873 mg/L in the virus-infested tomato experiment (Fig 7).
Conclusion
In the tomato expression system, thaumatin was able to be transcribed, translated and folded normally, which indicates that plant chassis do have an advantage over microorganisms in expressing proteins. 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 on transgenic production of thaumatin see BBa_K5160117 and BBa_K5160119.
Application Prospects
Transient expression is a rapid and efficient method for detecting protein expression that has been developed in recent years and is gradually being applied to research in all aspects of biology. In our project, transient infestation was applied to test the viability of chassis. Although transient expression is not used as the final means of thaumatin production, it is still a step that plants must undergo for heterologous expression.
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