Part:BBa_K5160117
pBWA(V)HS-35S promoter-thaumatin-3xHA-NOS terminator
Description
Transgenic technology is capable of introducing exogenous gene sequences into the genome of the host and thereby causing heritable changes in biological traits. As a result, it is often used to increase agricultural productivity, enhance crop resistance, improve the quality of agricultural products and protect the environment. The method has long been used not only as a traditional breeding method, but also as an important aid in the production of products in bioreactors. We decided to use transgenic means to introduce thaumatin gene into tomato callus tissues for heterologous expression using the integration ability of Agrobacterium Ti plasmid.
In order to provide a safe sweetener for people, our project hopes to come up with a method to produce thaumatin efficiently. Thaumatin comes from a tropical fruit in Africa, and is certified by the FDA and GRAS as a safe and reliable sugar substitute. After verifying that tomato chassis can express thaumatin correctly, the SZU-China 2024 team found a a new problem. For product use in our project, we need to further modify the production system to achieve efficient and stable synthesis of thaumatin in tomato.
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 BamHI site found at 1612
Illegal XhoI site found at 1929 - 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 1653
Illegal BsaI.rc site found at 1932
Usage and biology
Transgenic technology is based on the presence of a segment of T-DNA on the plasmid of agrobacterium. Agrobacterium inserts the T-DNA into the genome of the target plant after it enters the cell through wound invasion of the plant.
Agrobacterium Ti plasmid is a double-stranded covalently closed circular DNA molecule. It contains four functional regions, which are T-DNA region, Vir region, Con region and Ori region. The T-DNA region is the fragment that can be transferred and integrated into the plant genome; the Vir region encodes proteins that are involved in the processing and transfer of T-DNA; the Con region is related to the binding and transfer of the plasmid; and the Ori region is the plasmid replication initiation site.
When the plant body is damaged, the cells at the wound will secrete a large number of phenolic compounds. These phenolics can induce the expression of Vir genes in agrobacterium. Through chemotaxis, agrobacterium recognizes and attaches itself to the wounded site of the plant. The expression product of the Vir gene cuts off a single strand of T-DNA from the Ti plasmid to form a single-stranded T-DNA. Subsequently, the single-stranded T-DNA binds to the expression products of the Vir gene and the Con gene to form a complex. The complex transfers the T-DNA to the plant cell through contact between Agrobacterium and the plant cell. Upon entering the plant cell, the T-DNA is able to randomly integrate into the plant chromosome. This process can be single-copy or multi-copy, but is usually preferred near the coding or regulatory regions of a gene.
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.
3×HA tag：
This is a triple HA tag attached to the C-terminus of the protein, consisting of a 27-amino acid sequence, which is utilized for the purification and identification of the target protein.
In project design, to purify and identify the target protein while enhancing the detection signal and improving purification efficiency, we attach three HA tag proteins to the C-terminus of the target protein to create the corresponding vector. Subsequently, we introduce the target gene into the tomato genome sequence through transient infection technology or transgenic technology. When the target gene is expressed, the HA tag protein is also expressed, which can be used for subsequent Western blot (WB) detection and co-immunoprecipitation to verify the expression of the target protein.
NOS terminator：
A terminator on a plant expression vector that terminates the transcription of a gene.
Structural design
We first chose the eukaryotic promoter 35S for preliminary attempts of expression, and selected the binary vector pBWA(V)HS to construct the pBWA(V)HS_Thaumatin plasmid, which was transformed into Agrobacterium GV3101. To verify the success of 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 the target sequence.
DNA test:
We chose positive bacteria to infest the callus tissue, and when the callus tissue grew to a certain extent, it was inoculated and cultivated in rooting medium (Fig 5), and when the plant grew leaves, we picked the leaves to test whether the thaumatin gene was present in the genome of tomato. The results of the analysis showed that thaumatin DNA (719bp) could be detected (Fig 6).
Protein expression:
We collected leaf, flower and fruit samples, extracted proteins for WB assay and successfully obtained positive results around 28 kD and 10 kD, indicating successful expression of Thaumatin (28.36 kD) and Brazzein (9.75 kD) in transgenic micro-TOM (Fig 7). This is the first milestone in our experimental process, indicating that transgenic tomatoes with stable genetic ability to produce sweet proteins have been successfully bred.
Based on the immunoenzymatic ELISA for thaumatin, we concluded that the average level of transient expression of thaumatin under the expression of transgenic 35 promoter in tomato experiment was 11.1598 mg/L. (Fig 8).
Sweetness detection:
Improved ELISA for sweetness measurement
In order to use this principle to determine whether the sweetness protein in tomato can bind to the human receptor protein T1R2, we innovatively proposed a “double sandwich” method. Firstly, T1R2 is immobilized on a solid-phase carrier carrying an antibody against T1R2, and then a protein sample is added to allow Thaumatin to bind to T1R2, which is the key to our innovation. Next, we use an HRP enzyme-labeled secondary antibody to bind specifically to the already bound Thaumatin. Finally, we added the enzyme substrate TMB, which catalyzes a chromogenic reaction of the substrate, as a means of determining whether the sweet protein produced by tomato can bind to T1R2. As a control, we incubate a Thaumatin standard carrying the HRP enzyme as a secondary antibody on a solid-phase carrier and then use TMB for color development.
Based on this principle, we can give a judgment criterion: if the wells turn yellow with the addition of the substrate TMB, the Thaumatin in the sample can bind to T1R2, indicating that the protein has a sweet taste.
Our result allows us to determine whether the Thaumatin protein can bind to each other with the human-derived receptor protein T1R2 (Fig 9). However, our result from this method cannot reflect the sweetness index of the sample, this is because we cannot get the connection between absorbance and sweetness index. Finally, we were also unable to estimate the sweetness index of the tomato samples. Therefore, we further explored the sweetness detection method.
Electronic tongue detects sweetness
Before the test, we used the electronic tongue to establish our own sweetness model. Since the sweet protein Thaumatin is a protein substance rather than a common sugar, we referred to the concentration-response curve of sweeteners, conducted a large number of preliminary experiments, and set up Thaumatin tomato standard solutions with different concentration gradients (0%, 0.5%, 1%, 1.5%, 2%) to establish SVM sweetness models (parameters all set to 1, 2, 3, 4, 5).
We conducted electronic tongue detection on the control group tomatoes (i.e., uninfected wild Micro-Tom tomatoes), transgenic tomatoes with the 35S promoter, and transgenic tomatoes with the E8 promoter. Subsequently, the taste characteristics detected by the electronic tongue were input into the SVR model for analysis. According to the model prediction analysis and calculating the average value of the data, the prediction result of the wild type is 0.0109, indicating that the tomato sample of the control group is equivalent to the standard Thaumatin tomato solution with a concentration of 0 ppm, further demonstrating the usability of our model. The result of transgenic tomatoes with the 35S promoter is 21.6013, that is, the Thaumatin contained therein is equivalent to the standard Thaumatin solution with a concentration of 21.6013 ppm. By the same reasoning, it can be concluded that the Thaumatin in tomatoes with the E8 promoter is equivalent to the standard Thaumatin solution with a concentration of 21.6040 ppm.
Subsequently, according to the concentration-response relationship curve of Thaumatin established by Grant E. DuBois and D. Eric Walters, it can be calculated that the sweetness level of Thaumatin in transgenic plants containing the 35S promoter is 8.65, and the sweetness level of Thaumatin in transgenic tomatoes containing the E8 promoter is also 8.65.
Finally, according to the calculation based on the concentration-sweetness level of sucrose, it can be known that Thaumatin in transgenic tomatoes is equivalent to the standard sucrose solution with a concentration of 8.65%.
Conclution
We constructed the gene sequence of 35S promoter-thaumatin-3×HA-NOS terminator on pBWA(V)HS_Thaumatin plasmid and integrated it into the genome of Agrobacterium GV3101 by using an expression vector, and subsequently transgenicized it by using Agrobacterium GV3101 to infiltrate tomato callus tissues. We cultivated the callus tissues into mature plants, and we verified the successful expression of the protein thaumatin in tomato by DNA assay and WB assay. What's more, we concluded in the thaumatin concentration and sweetness assay that thaumatin expressed in tomato fruits had the correct protein fold, which indicated the correctness of our selection of tomato.
On the other hand, we performed phenological comparisons and metabolic fraction analyses between transgenic tomatoes and wild-type plants. The results showed that heterologous expression of thaumatin does not adversely affect tomatoes.
Application Prospects
In our final design, thaumatin can be obtained directly from tomato juice without purification. This sweetener is safe and reliable, does not produce calories, and is able to satisfy people's sweet taste needs. In the future, thaumatin produced from tomato can be used in various food applications instead of sugar.
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