Overview

With the continuous improvement of living standards, people have higher demands for food health and safety. SZU-China 2024 innovatively creates a new tomato-based sweetener production system. In our project, we adopt the approach "Fruit+Biopower", integrates two key components: the Expression System and the Localization-Storage System. Together, they enable the production of Thaumatin in tomato fruits, offering a novel dietary solution for diabetics and those struggling with obesity, while enriching the world of sweeteners.

-Efficient and Stable Expression of Thaumatin : The Expression System optimizes both the chassis and genetic pathways, allowing for the heterologous, efficient, and stable expression of Thaumatin in tomatoes.

-Increasing the Storage of Thaumatin : The Localization-Storage System acts as a "GPS" for the sweet protein, facilitating its targeted positioning and storage within the fruit.

By combining these two systems, we have, for the first time in iGEM history, successfully modified tomato fruits as biofactories for sweetener production. This breakthrough offers the public a novel “space station” for artificial sweetener Expression, bridging the gap between sugar control and flavor enjoyment.

Expression System

In the Expression System, we successfully established sugar production systems in both prokaryotes and plants. Among various kinds of sweet proteins, Thaumatin (BBa_K5160003) and Brazzein (BBa_K5160004) stood out from the others due to their strong thermal stability and acid resistance. Thaumatin is derived from the aril of the African plant Thaumatococcus daniellii (Benth), while Brazzein comes from the pulp of Pentadiplandra brazzeana Baillon (P. brazzeana), which grows in the African rainforest. Both proteins bind to sweet taste receptors T1R2 and T1R3 on the human tongue, inducing the perception of sweetness. Additionally, they can be fully digested by human body, breaking down into common amino acids with little to no caloric production. Given these benefits, we proceeded with experimental validation for both sweet proteins.

Expression in Prokaryotic

1. For the prokaryotic expression of Thaumatin, we constructed the pET28a-T7 promoter-6× His-Thaumatin-6× His-T7 terminator (BBa_K5160112) plasmid in Escherichia coli BL21(DE3), thus achieving heterologous protein expression. These include the T7 promoter(BBa_R0187), T7 terminator (BBa_M50060), and 6x His (BBa_K157011) tags.

2. For the prokaryotic expression of Brazzein, we constructed the pET28a-T7 promoter-6× His-Brazzein-6× His-T7 terminator (BBa_K5160111) plasmid in Escherichia coli BL21(DE3), thus achieving heterologous protein expression. These include the T7 promoter(BBa_R0187), T7 terminator (BBa_M50060), and 6x His (BBa_K157011) tags.

3. The T7 promoter is a strong promoter derived from T7 phage, widely used in molecular biology and biotechnology for gene expression control. It specifically responds to T7 RNA polymerase, allowing precise regulation of target gene expression. The T7 terminator is a specific DNA sequence that halts transcription, ensuring the newly synthesized RNA is properly released from the transcription machinery. Finally, the 6x His tag is commonly used for the purification and detection of recombinant proteins, as it does not interfere with the protein's structure or function. By attaching the 6x His tag(BBa_K157011) to the C-terminal of the proteins, we enhanced detection accuracy and minimized false negatives during experimental assays.

Expression in Tomato

We subsequently verified the expression of Thaumatin and Brazzein in tomatoes using both transient expression technology and transgenic technology.

Ⅰ. TRV verification

Transient infection is an effective method for quickly testing the functionality of plant expression vectors, with results obtainable in a short period. Studies have shown that transient infection can be achieved through leaf injection of viral vectors. To save time, we utilized viral transient infection technology to verify expression in tomatoes.

1. In this part, we used TRV virus as a vector for transient infection. TRV viruses contain two RNA strands called TRV1(BBa_K5160007) and TRV2. We inserted the target protein sequence into TRV2 and successfully expressed the target protein heterogeneously in tomato with the help of TRV1.

2. Considering plants' antiviral defense mechanism, gene silencing (siRNA production), which degrades viral RNA, we included P19 (BBa_K5160006), a viral silencing suppressor. P19 binds to siRNA, preventing the degradation of viral RNA and allowing the TRV virus to successfully replicate and spread in tomato cells. To ensure experimental accuracy, we also constructed the plasmid pTRV2-35S promoter-P19-NOS terminator (BBa_K5160116).

3. Additionally, we utilized the 35S promoter (BBa_K788000) and the flag tag. As a strong promoter, 35S promoter can induce target gene expression in any tissue at any time without spatio-temporal specificity. Flag can complete the detection of the target protein. By using 3x flag(BBa_K5160010) protein tag, we can improve the accuracy of detection.

4. Finally, we use red fluorescent protein mCherry(BBa_K5160005) to monitor the effectiveness of viral vectors. For the accuracy of the experiment, we also constructe the blank control plasmid pTRV2-35S promoter-mCherry-NOS terminator (BBa_K5160115) for mCherry. Based on this, we constructed pTRV2-35S promoter-Thaumatin-3× Flag-NOS terminator (BBa_K5160113) and pTRV2-35S promoter-Brazzein-3× Flag-NOS terminator (BBa_K5160114) plasmid, and achieved initial validation in tomato using transient infection technique.

Ⅱ. GMO Whole Strain Expression

We then validated the system in tomatoes using transgenic technology. By introducing foreign gene sequences into the host genome, we induced heritable changes in biological traits. In this phase, we continuously optimized the genetic pathway and incorporated new components, ultimately achieving stable expression of Thaumatin and Brazzein in tomatoes.

1. We replaced the promoter with a tomato fruit ripening specific promoter E8 (BBa_K5160002). As a specific promoter, E8 can express Thaumatin specifically in tomato fruits and reduce the metabolic burden of roots and leaves.

2. At the same time, we used the protein tag HA (BBa_K5160012) commonly used in eukaryotes. Since it does not interfere with the structure and function of the target protein, it is often used to detect, isolate and purify proteins. In addition, we used a 3x HA(BBa_K5160011) tag attached to the C-terminal of the protein to improve the accuracy of detection and reduce false negative results in the experiment.

And finally, we built pBWA(V)HS-35S promoter-Thaumatin-3× HA-NOS terminator (BBa_K5160117) and pBWA(V)HS-35S promoter-Brazzein-3× HA-NOS terminator (BBa_K5160118) control plasmid. pCAMBIA1301-E8 promoter-Thaumatin-HA-NOS terminator (BBa_K5160119) and pCAMBIA1301-E8 promoter-Brazzein-HA-NOS terminator were constructed (BBa_K5160120) experimental plasmid. These constructs were validated in tomatoes using transgenic technology, and results confirmed that the E8 promoter significantly enhances the tomato's ability to heterologously express the protein.

So far, we have completed the iteration of Thaumatin heterologous expression in different chassis. For more information, see Engineering.

Localization-storage System

Considering that protein degradation may lead to a decrease in protein content, we constructed a localization-storage system and constructed the pGD-35S promoter-SPS-NTPP-Thaumatin-EGFP-NOS terminator (BBa_K5160121) plasmid. The sweet protein Thaumatin is specifically stored in vacuoles to increase its concentration.

1. Our localization-storage system (BBa_K5160121) is based on the SPS-NTPP vacuole signal peptide targeting system.

2. SPS-NTPP sequence of sweet potato spore N-terminal propeptide (BBa_K5160009). It is a vacuole-localization protein derived from sweet potatoes. By adding SPS-NTPP to the N-terminal of Thaumatin, we are able to precisely target the sweet protein into the vacuole for storage.

3. EGFP (BBa_K4251013) is a highly fluorescent protein. By using confocal microscopy to track and analyze the location and aggregation of the fluorescent focus of EGFP, we can determine whether SPS-NTPP is able to target the protein to the vacuole. At the same time, for the accuracy of the experiment, we also constructed EGFP blank control pGD-35S promoter-Thaumatin-EGFP-NOS terminator(BBa_K5160122) plasmid for comparison with the experimental group.