Prologue

This year, SZU-China will introduce a healthier and more natural sweet sauce product, "Sweetein," to everyone. After a series of experiments, we have successfully obtained transgenic tomatoes for the expression of sweet proteins and verified their expression efficacy. On this page, we will present to you the continued product upgrades, iterations, and corresponding experimental plans.

Targeted storage of sweet proteins in tomatoes

In our project to genetically modify Micro-Tom tomatoes to produce the sweet protein Thaumatin, we aim for successful expression and targeted aggregation of the sweet protein into the vacuoles of fruit cells for storage (for details, see Outline)^[1]. Therefore, we plan to conduct a proof-of-concept test on our transgenic Micro-Tom.

At the end of the Results page, we used tobacco leaves as experimental material and preliminarily demonstrated that our SPS-NTPP targeting peptide can guide sweet proteins to the vacuoles in tobacco cells, indicating that the plasmid construction idea of SPS-NTPP-Thau-EGFP is feasible and that the targeting peptide ^[2] itself is not problematic. However, differences in the chassis may also affect the function of the targeting peptide. Therefore, we intend to repeat the tobacco confocal experiment when verifying in tomatoes and if the results show that the targeting peptide can play its role correctly, we will also conduct further tests on the impact of the targeting peptide on tomato metabolism and its targeting efficiency.

Currently, we have completed the Agrobacterium infection and confocal experiments on tomatoes. The results show that the location of green fluorescence (where the sweet protein is) avoids the location of red fluorescence (autofluorescence of chloroplasts), indicating that SPS-NTPP can also guide sweet proteins to the vacuoles in tomatoes (Fig 1).

Fig 1. Confocal microscopy results image of tomato. red:ChlorophyII autofluorescence(to indicate cytoplasmic location), green:EGFP

Our ultimate goal is to store sweet proteins in the vacuoles of fruit cells to indirectly increase their expression levels by reducing degradation. To verify this hypothesis, we plan to continue with experiments, constructing the Thau-EGFP_pGD plasmid and introducing it into tomatoes as a control group, while the experimental group will still be introduced with the SPS-NTPP-Thau-EGFP_pGD plasmid. Subsequent confocal microscopy will be used to count EGFP foci in cells for comparative verification. Additionally, after team discussion, we also plan to perform mass spectrometry identification to explore whether the use of the SPS-NTPP targeting peptide will have an impact on the tomato's own metabolism.

If the outcomes are favorable, we plan to incorporate the SPS-NTPP targeting peptide into our future products to ensure the production of sweet proteins, offering a unique transgenic tomato from SZU-China as an efficient and natural platform for sweet protein production!

Product Design and Advancement

Our HP team members observed during a visit to the SIAL food exhibition that many companies offer their products in packaging that comes in more than one size. We interviewed some vendors about their product philosophies and found that the use of different package sizes aims to meet the needs of a broader audience. For individual consumers, there is a preference for smaller, more portable packaging options^[3], while factories or shops often opt for larger sizes to reduce costs on retail packaging and enhance the cost-effectiveness of the raw materials. Subsequently, our team visited the Yuanqi Forest company and noticed that many of the raw materials they use are in concentrated forms, which also reduces costs to a certain extent.

In our team meeting, we reached a consensus that the production of a product must consider the needs of its target audience. Consequently, we have envisioned our product to be produced in the form of tomato sweet puree, and we plan to employ drying technology to concentrate it for different usage forms and audiences, ultimately yielding a series of sweet puree products named "Sweetein" (Fig 2). On this page, we will validate the feasibility of producing Sweetein and the ease of its processing.

Fig 2. Tomato sweet puree product

Our products are divided into three different levels of sweetness, corresponding to the use of sweet protein tomatoes of different weights as raw materials. This means that products of different sweetness levels contain different equivalents of sweet proteins and therefore have different uses(Fig 3).

Fig 3. The corresponding sweet protein equivalents for different sweetness levels (the sweet protein equivalents in the picture serve only as a reference, please refer to the actual product for specifics).

Fig 4. Possible product forms of sweet pulps of grades A, B, and C.

Next, we conducted a simple simulation of the production process (The production process is for reference only).

Fig 5. A,B. Use a juicer to crush the fruit. C,D. Filter our tomato pulp.

Fig 6. Left and middle: Put the obtained clarified liquid into a dryer for concentration and drying. right: Package the concentrated sweet pulp.

First, tomatoes are crushed and then filtered. The resulting juice becomes Grade A sweet pulp. Subsequently, the materials for Grade B and Grade C sweet pulp are placed into a dehumidifying dryer for dehydration treatment. According to the required sweetness level, they are concentrated to different multiples of concentrated liquid. Finally, the end product is obtained (Fig 5,6).

Through this simulated experiment, we have verified the feasibility and simplicity of producing Sweetein sweet pulp. By adopting a series of straightforward operations, we are able to produce healthy Sweetein sweet pulp with rich nutritional value. Undoubtedly, this reduces production costs and enhances the cost-effectiveness of the product. In the future, we plan to extend this technology to more crops and products, aiming to create a world of low-calorie sweetness.