Part:BBa_K5160119
E8 promoter-thaumatin-HA-NOS terminator
Overview
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 2163
Illegal NotI site found at 2200
Illegal NotI site found at 2302 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2825
Illegal XhoI site found at 3142 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2196
Illegal NgoMIV site found at 2362 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2866
Illegal BsaI.rc site found at 3145
Usage and biology
In the conventional transgenic pathway, synthesis of thaumatin and brazzein initiates expression under the CaMV 35S promoter. However, the CaMV 35S promoter lacks stability. On the one hand, a large number of studies have shown that although the CaMV 35S promoter is highly expressed, it affects the expression of neighboring genes, leading to disease in plants. On the other hand, the activity of CaMV 35S promoter varies in different tissues and cells of plants, which makes it difficult to ensure the expression level.
In order to ensure the stability of sweet protein expression in transgenic tomato, we need to optimize the promoter. Therefore, we chose the tomato fruit-specific promoter E8 instead of the 35S promoter. This tissue-specific promoter can not only accumulate the expression products of target genes in certain organs or tissue sites and increase regional expression, but also avoid unnecessary waste of plant nutrition.
E8 promoter:
The E8 promoter has a highly specific expression pattern in tomato fruit. It is mainly activated at specific stages of tomato fruit development, driving the preferential expression of downstream genes in fruit tissues. The E8 promoter is significantly more active in fruit compared to other tissues such as roots, stems and leaves. It can be utilized to achieve precise regulation of specific genes in fruits. By linking the target gene to the E8 promoter, the target gene can be expressed only in tomato fruits, avoiding unwanted effects in other tissues. This is important for improving the quality and characteristics of tomato fruits.
During fruit ripening, the E8 promoter can initiate the expression of a series of genes that are involved in physiological processes such as fruit color change, texture softening, and flavor substance synthesis. These changes are the result of the mutual regulation of the E8 promoter and ethylene.
There are at least two major regions in the E8 promoter sequence that contribute to its transcriptional regulation. One is the upstream region from -2181 to -1088, which contains ethylene-responsive transcriptional elements. When ethylene concentration is elevated, the expression of the E8 promoter and ethylene synthesis genes increases simultaneously, forming a positive feedback regulatory mechanism that further promotes fruit ripening. And in the absence of ethylene synthesis, another region comes into play. The sequence from -409 to -263 of the transcription start site is sufficient for ripening-specific transcription. For more information about this part, please see BBa_K5160002
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.
3xHA 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.For more information about this part, please see BBa_K5160011
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.For more information about this part, please see BBa_P10401
Structural design
We selected the binary vector pCAMBIA1301 to construct the pCAMBIA1301_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
Transformation success
We used specific primers for pcr and proved the success of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis (Fig 4). Then the callus tissue was infested with positive bacteria, and when the callus tissue grew to a certain extent, it was inoculated in rooting medium (Fig 5).
Expression at DNA level
The callus tissues were cultivated on rooting medium, and after the plants grew leaves, we picked the leaves to test whether our genes were present in the genome of tomato. The results of the analysis showed that all of thaumatin's DNA could be detected (Fig 6).
Expression at protein level
We harvested the leaves, flowers and fruit samples and extracted proteins for WB assay. Eventually, we only succeeded in detecting the presence of a positive band around 28 kD in the fruit, but not in the leaves and flowers (Fig 7). This result suggests that Thaumatin (28.36 kD) was successfully expressed in our transgenic tomato under the initiation of the E8 promoter.
(A) Protein was examined in transgenic tomato leaf with the E8 promoter.
(B) Protein was examined in transgenic tomato flower with the E8 promoter.
(C) Protein was examined in transgenic tomato fruits with the E8 promoter.
We analyzed the efficacy of the E8 promoter to express proteins. We determined the concentration of thaumatin expressed with the 35S promoter and the E8 promoter in transgenic tomatoes by immunoenzymatic assay (ELISA). By comparison, we obtained that the average level of thaumatin expressed under the induction of the constitutive promoter 35S was 11.1598 mg/L; while the expression level using the fruit ripening-specific promoter was 11.0591 mg/L. (Fig 8). There was no statistically significant difference, indicating that the expression level of the fruit-specific promoter E8 had reached the expression level of the strong promoter 35S. Compared with 35S, the E8 promoter not only ensures fruit-specific expression and avoids burdening other parts of the tomato plant, but also ensures the high expression level of thaumatin.
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
Model construction
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).
SVM analysis of the electronic tongue for Thaumatin tomato standard solutions of different concentrations shows that the first principal component accounts for 93.63% of the information in the PCA-SVM, and the second principal component accounts for 3.14% of the information. It can be seen that the total contribution rate of the first principal component and the second principal component is 96.77%. The R-squared value in the established SVM curve reached 0.9839, indicating that the model is good and usable.
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%.
Conclusion
Based on the above results, it can be concluded that our designed E8 promoter has shown excellent specific expressions and we can ensure that thaumatin is efficiently expressed only in fruits. In the future, there is an opportunity to industrialize this production model.
Reference
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