Overview

The goal of scientific research is to understand the world and transform it. Do we have the ability to construct new and efficient biological systems through molecular design, so as to realize the artificial synthesis and reconstruction of complex regulatory pathways, in order to meet the major challenges in the fields of biofuels, drug production, environmental remediation, agricultural improvement and disease treatment? This question has become a cutting-edge topic in synthetic biology and life sciences in general.

As the global population continues to grow, the demand for yield and quality of crops, food, natural products and medicines continues to increase^[1]. Through the technology of plant synthetic biology, innovative solutions can be designed and optimized for plant systems to enhance food production, increase crop resilience, and promote sustainable production of renewable energy, pharmaceuticals, and natural products.

The use of sugar substitutes is becoming more common as concerns grow about the health risks associated with excessive sugar intake. However, recent studies have identified significant safety issues with many common substitutes. To address this issue, SZU-China innovatively proposed the use of plant synthetic biology to develop safe and effective sugar substitutes.

However, compared with unicellular organisms with simplified chromosome structures and relatively simple regulatory mechanisms, multicellular organisms (plants e.g.) with complex genome structures and extensive and fine regulatory networks have limitations in their efficiency as biosynthesis factories, and it is difficult to replace traditional means of microbial fermentation in practical applications. The main challenges facing plant synthetic biology lie in the three areas of component development, gene circuit design and biological device construction.

1. How to achieve rapid validation of components?

2. How to regulate the spatio-temporal specific expression of plants?

3. How to ensure efficient storage of products?

In response to these issues, our project creatively utilizes the inherent properties of plants to propose synthetic biology methods suitable for plant platforms based on traditional synthetic biology:

1. Using plant viral vectors for rapid validation of component functions.

2. Utilizing endogenous plant components to achieve spatio-temporal specificity in product expression.

3. Targeted storage of products using protein sorting mechanisms

Based on the above three approaches, we built an efficient and stable production line in tomato and realized the rapid functional identification of Thaumatin as well as the spatio-temporal expression control and efficient storage in plants. Our work is constructive for the improvement of plant synthetic biology, and we hope it can provide more data and reference value for the development of plant synthetic biology.

Through experimental validation, our designed Thaumatin production system successfully operates in tomatoes. The genetic transformation methods, expression systems, and localization-storage systems have demonstrated excellent capabilities in tomato fruits, and these experimental results confirm the feasibility and potential of our design. Looking back at the outstanding projects in plant synthetic biology from iGEM between 2010 and 2022, our project is the first one to efficiently utilize tomato fruits for spatio-temporal specific production in the iGEM competition. This initiative will inspire future teams to achieve practical applications in the field of plant synthetic biology.

How to achieve rapid validation of components?

The long growth cycle of plants and the often long period validation of links between genotypes and phenotypes are plant-specific challenges that must be overcome by teams conducting plant programs^[2]. Over the 20 years of iGEM in the field of plant synthetic biology, various teams have contributed their innovative ideas. The Nevada team in 2010 (United States) used Nicotiana tabacum cells for concept validation. As a rapidly growing model organism, Nicotiana tabacum significantly shortened the experimental validation time. Meanwhile, the Cambridge-JIC team in 2016 (UK) developed a chloroplast transformation toolkit that reduced the time required for homoplastid transformation from several months to 1-2 weeks. This year, SZU-China, building on the work of predecessors, has innovatively proposed our own solution—utilizing viral vectors to deliver target components into plants, providing a powerful development tool for the iGEM community.

Transient expression technology is a method for rapidly analyzing gene function in plant and animal cells. It achieves high-level expression of target genes in a short time by introducing exogenous genes into host cells. The traditional approach for transient expression involves soaking plant leaves in Agrobacterium. However, in our practical application, we found that this method struggles to maintain traits. More troubling is that it is difficult to achieve infection in other plant organs. This means that traditional transient expression methods are challenging for effective characterization in plants, let alone serving as a powerful tool for other iGEM teams.

Therefore, on this basis, our project proposes an efficient genetic transformation method that can be applied to transient expression to rapidly characterize the function of components. We utilized viral expression vectors for validation. Viral expression vectors are derived from plant viruses with double-stranded RNA. They are reverse transcribed into cDNA in vitro by the action of reverse transcriptase. The cDNA is specifically modified and cloned to turn it into a vector that is non-toxic but has the ability to self-replicate and express exogenous genes. When we load the target sequence onto the vector and inject the viral vector into the plant, the viruses will self-replicate in the plant and characterize the viability of the target sequence in the process. They can exist in the plant for a longer period of time and can be transferred within the plant, which facilitates us to test them in different parts of the plant. More importantly, this method can quickly achieve the results of the test and play a great role in pre-experimentation. Using this transient expression method, we can greatly shorten the time of component characterization, accelerate the screening and determination of components, and provide a convenient means of verification for plant synthetic biology researchers(Fig 1).

Fig 1. Transient expression mediated by TRV virus

For the validation of this panel, our project used the TRV virus as a vector for transient invasion. TRV virus contains two RNA strands, TRV1 (BBa_K5160007) and TRV2. We inserted the target protein sequences on TRV2 and constructed a sweet protein expression plasmid pTRV2-35S promoter-thaumatin-3× Flag-NOS terminator (BBa_K5160113) by utilizing a strong promoter, the CaMV 35S promoter (BBa_K788000), and a 3x Flag (BBa_K5160010) protein tags to construct expression plasmids for the sweet proteins pTRV2-35S promoter-thaumatin-3× Flag-NOS terminator (BBa_K5160113) and pTRV2-35S promoter-brazzein-3× Flag-NOS terminator (BBa_K5160114) and successfully heterologously expressed the target proteins in tomato with the assistance of TRV1(Fig 2).

Fig 2. Plasmids used for transient expression of Thaumatin and Brazzein.

Meanwhile, considering that plants have gene silencing, a mechanism of antiviral defense, which can produce siRNA to degrade viral RNA. To prevent the effect of plant gene silencing on the experiment, we used P19 (BBa_K5160006), a silencing repressor of the virus. It can bind to siRNA and prevent the binding and degradation of viral RNA, enabling the TRV virus to replicate and spread smoothly in tomato cells. Thus, we completed the validation of the transient infiltration technique in tomato to achieve the genes and components, which provides guidance for the subsequent optimization of the pathway.

How to regulate the spatio-temporal specific expression of plants?

In traditional microbial fermentation, bacteria or yeast express the product as an intact individual, and the expression of the product can be manipulated by regulating the overall level of the community. However, in plant factories, due to the complex tissue and organ structure of plants, the expression of products by an intact plant body has a huge impact on the physiology and metabolism of the plant. Therefore, we need to regulate the spatial and temporal expression of the products to ensure that the plant body maintains normal growth and development while efficiently expressing the products, which has become a difficult problem in the study of plant synthetic biology.

Our project this year proposes an idea to solve this problem by designing regulatory parts from the complexity of metabolic pathways and structures in the plant itself to achieve spatio-temporal specific expression of the products.

Time-specific regulation in plants

During plant growth and development, the expression of different genes will be precisely regulated at specific time nodes to adapt to the needs of different stages. Time-specific regulation can be realized in a variety of ways, such as the activation of specific promoters at different developmental stages, the role of transcription factors at specific times, and the conduction of hormonal signals at specific moments^[3]. Such regulatory mechanisms ensure that plants are able to perform critical physiological activities at the right time, thus better adapting to environmental changes and ensuring survival and reproduction. Therefore, our project decided to use plant time-specific promoters to control the expression of products at the right stage(Fig 3).

Fig 3. Tomato fruit development regulated by ethylene.

Space-specific regulation of plants

Plant growth and development require the rational allocation of limited resources. Space-specific regulation enables plants to allocate energy, material and metabolic resources in a targeted manner according to the needs of different parts of the plant. This helps to avoid wasteful and ineffective allocation of resources and improves the overall survival competitiveness of plants^[4]. In particular, fruits, as the reproductive organs of plants, usually accumulate a large amount of nutrients and metabolites. Therefore, our project utilizes space-specific promoters to achieve precise control of the expression of target products, so that the products can be stably expressed in the fruit without affecting other parts(Fig 4).

Fig 4. Schematic representation of fruit intentionally expressed from the E8 promoter.

Function of E8 promoter

Considering the practical application in our project, we hope that the sweet protein produced can eventually reach the market to benefit more consumers. As an edible protein, Thaumatin expression in fruits not only helps to maintain taste, but also reduces safety risks and purification costs. Therefore, we chose the fruit-specific E8 promoter to express Thaumatin. As a fruit-specific ripening-inducible promoter, the E8 promoter possesses both temporal and spatial specificity.

1. As one of the most widely characterized tomato fruit ripening-specific promoters, E8 promoter shows strong conservation among different tomato varieties. Therefore, it can realize the specific expression of thaumatin within tomato fruits without affecting the normal work of roots and leaves.

2. E8 promoter, as an ethylene-regulated promoter, this class of specific elements can play a role in stabilizing the expression of exogenous genes.

In order to verify the specificity of the E8 promoter in initiating the expression of downstream genes in fruits, we constructed two plasmids, E8 promoter-Thaumatin-1x HA (BBa_K5160119) and E8 promoter-Brazzein-1x HA (BBa_K5160120), and introduced them into Agrobacterium GV3101 respectively to complete the invasion of Micro-Tom callus tissue. The leaves, flowers and fruits were sampled for protein extraction at the appropriate time after transplantation and WB tests were performed to evaluate the performance of the E8 promoter. The results showed that the samples from the E8 group did not express protein in the leaf and flower experiments, whereas clear bands could be seen in the Ca MV35S group. On the contrary, in the fruit WB experiments, the samples from the 35S group and the E8 group showed bands, but the E8 bands were darker(Fig 5).

Fig 5. Western blot results from E8 promoter transgenic tomato leaves and flowers. Western blot results for Thaumatin leaf (A), Thaumatin flower (C), Brazzein leaf (B) and Brazzein flower (D), Thaumatin fruit (E), Brazzein fruit (F).

The results of these experiments indicate that the E8 promoter can correctly function as a fruit-specific promoter in our project to produce sweet protein from transgenic tomatoes(Fig 6).

Fig 6. Growth of the transgenic tomato.

The results of these experiments not only support our project, but also have implications for other teams. Our work has led to what promises to be a more widely known and commonly used synthetic biology component.

How to ensure efficient storage of products?

Various proteases are present in plant cells that may degrade expressed proteins. In addition, environmental factors such as changes in temperature and humidity may accelerate the degradation of proteins. This can lead to a gradual decrease in the amount of stored proteins and affect their availability^[5]. This is the main reason why mass production of plant chassis is not possible. To address this issue, our project utilizes the subcellular compartments of the plant to protect the proteins from being affected, and specifies the destination of the proteins after production has been achieved, prompting the proteins to accumulate within the compartments(Fig 7).

Fig 7. (A) sorting mechanism of PSV and (B) sorting mechanism of LV.

Subcellular compartments

Plant cells consist of multiple membrane-separated organelle compartments, each of which has evolved to optimize and undertake specific biochemical functions. Proteins are carefully choreographed through an assembly line of the nucleus, ribosomes, endoplasmic reticulum, and Golgi apparatus, where they are transcribed, translated, and folded so that they arrive at the right compartment through the right route, at the right time and in the right structure. It can be seen that the subcellular composition of the "circuit" gives the plant production system automated characteristics, only need to add a specific "GPS" signal to the product, they can automatically localize to a certain compartment.

Advantages of vacuole localization

In our project, Thaumatin is a soluble protein with acid stability^[6]. In terms of contents, the liquid environment inside the vacuoles is acidic in pH, which is suitable for Thaumatin storage. In terms of properties, plants form special vacuoles containing large amounts of specific substances in specific tissues, storage vacuoles and lysis vacuoles. Tomato fruits and seeds belong to storage tissues, and the nature of their vacuoles is closer to that of storage vacuoles. Therefore, the vacuoles within the fruits contain less hydrolytic enzymes and are more suitable for Thaumatin storage. From the metabolic aspect, tomato as a berry, during fruit ripening, complex processes such as the transformation of chloroplasts to chromoplasts, softening of cell walls, pigment accumulation, and changes in hormone levels occur in the cells, and at this time the cytoplasmic fluid environment is complex and unfavorable for the stable existence of Thaumatin. On the contrary, the environment inside the vacuole is in a relatively stable state, therefore, the vacuole compartment can prevent the influence of the huge environmental changes on Thaumatin during the fruit ripening process.

Function of SPS-NTPP

We chose to use the sweetpotato sporamin-N-terminal prepeptide SPS-NTPP sequence (BBa_K5160009) for fusion expression with the sweet protein sequence. SPS-NTPP is a vacuolar signaling peptide derived from sweetpotato tuberous roots, which targets and stores sporin to the vacuole. After the protein completes processing in the endoplasmic reticulum, it is directed to the Golgi apparatus for further processing via COP II, followed by targeting to the vacuoles via the SPS-NTPP vacuolar sorting determinants (VSDs), binding to the specific vacuole sorting receptor (VSR), and subsequently transferring the protein to the vacuoles for storage, and after the targeting is completed the SPS-NTPP is able to self-decompose without any effects(Fig 8).

Fig 8. Principle of localization of SPS-NTPP

In order to verify the function of the localization peptide, we constructed pGD_SPS-NTPP-Thaumatin-EGFP plasmid (BBa_K5160121), which was introduced into Agrobacterium tumefaciens and infested the plants, and the positional distribution of fluorescence focuses and aggregation were compared with the help of confocal microscopic observation. The results showed that SPS-NTPP possessed significant ability to localize vacuoles. This suggests that vacuole localization may be an effective solution to the lack of plant production yield(Fig 9).

Fig 9. Confocal microscopy results image of tomato. red:ChlorophyII autofluorescence(to indicate cytoplasmic location), green:EGFP

Our contribution to Plant Synthetic Biology

1. Virus-mediated transient expression: a rapid method to validate components

Virus-mediated transient expression systems can quickly and effectively validate the function of various components in plants. Through this method, the effects of specific components on plant gene expression, metabolic pathways, and physiological functions can be observed in a relatively short period of time. It provides researchers with a convenient means to screen and characterize components with specific functions, enables rapid assessment of component activity and specificity, and contributes to a deeper understanding of plant gene regulatory networks and metabolic mechanisms. This year we propose a viral vector TRV that can be used for rapid validation of tomato chassis, which will provide a feasible method for future teams when conducting plant research.

2. E8 promoter: spatial and temporal specificity of regulation

As a promoter with spatial and temporal specificity, the E8 promoter can precisely control the expression of target genes in specific time and space. In the temporal dimension, it can initiate the expression of target genes at the right time according to the plant's growth and development stage, ensuring that the genes function when they are most needed. In the spatial dimension, the E8 promoter can specifically activate the target gene in a particular tissue or organ, avoiding non-essential expression in other parts. This year, our project used the E8 promoter for the first time in the history of iGEM. Through our experimental characterization, we verified that the E8 promoter is characterized by high expression specificity and high expression level, and our work is expected to provide an efficient and stable synthetic biology part in plants for future iGEM teams.

3. SPS-NTPP: ensure efficient product storage

We provide a safe, stable and efficient storage environment for specific products synthesized by plants through a series of fine regulatory mechanisms. At the structural level, SPS-NTPP utilizes specific localization signals to accurately locate and isolate the products for storage, preventing them from being interfered and damaged by external environment factors and other metabolic processes within the cell. From the perspective of metabolic regulation, it can coordinate the metabolic network of the plant, dynamically adjust the storage capacity and mode according to the synthesis rate of the products, and ensure that the products will not burden the normal physiological metabolism of the plant during the accumulation process. For the field of plant synthetic biology, SPS-NTPP provides a reliable guarantee to improve the yield and quality of the target products, and opens up a new pathway for other teams to develop plant products with high value-added in the future as well as to realize sustainable agricultural development.

How to think about plant-related issues?

In this year, SZU-China innovatively used tomato as a production chassis of Thaumatin to achieve spatio-temporal specific production of tomato fruits for the first time in the history of iGEM. During our exploration this whole year, we have experienced many dilemmas and challenges facing plant synthetic biology. As a result, we are even more aware of how important a guiding instrument on plant synthetic biology is for project advancement. Therefore, we have compiled our questions related to plant synthetic biology and how we should think about carrying out a plant synthetic biology project throughout the year, hoping to provide inspiration and advice to iGEM teams who are interested in this topic, and to help them better build their own projects.

Stage 1. Background research

Carefully read the official iGEM webpage about plant synthetic biology. There, you can see many interesting plant synthetic biology designs, many of which have made great contributions to the field of plant synthetic biology. By reading this website, you can get inspiration for your project and thus start your exploration of plant synthetic biology.

Stage 2. Optimization of effects

1. Carefully consider the plant chassis chosen. The plant chassis is crucial to the whole project design, not only for the subsequent access design, but also for how to complete the verification expression. Therefore, we suggest to consider the background of the project and the actual situation, and carefully consider the selected plant chassis.

2. Rational selection of biological components. Biological components are crucial for plant synthetic biology. A good biological component can do twice as much with half the effort and bring great surprises. Therefore, we suggest to start from the plant itself and explore the components available in the plant.

3. Design gene pathways carefully. Considering that there are many complex metabolic pathways in the plant itself, our modification of the plant may affect the whole body. Therefore, we should be careful in designing gene pathways to ensure that our design will not affect the plant itself.

4. Comprehensively consider the safety issue. Plants play a very important role in nature, so modification of plants may bring some negative impacts on the ecological environment. Therefore, we sincerely suggest that we should consider all possible safety issues and formulate relevant preventive strategies during the whole project phase.

Stage 3. Normative Guidelines

1. If possible, we suggest that we listen to the suggestions of many stakeholders and conduct field visits during human practice. After taking into account the actual situation, we would like to optimize and improve the project and form a guideline to guide the subsequent team research.

2. If possible, we would like to document the challenges we have faced in plant synthetic biology and our solutions. This is not only a legacy of plant synthetic biology, but also builds a bridge of good mutual support in the iGEM community. Through this record, we can provide inspiration and build a better world of plant synthetic biology for other teams interested in plant synthetic biology.