Results

• Research and Analysis of Basic Parts: Conducted literature research to obtain the target sequence and performed protein structure alignment analysis.
• Construction of Composite Parts: Successfully assembled 13 expression vectors and created new composite expression elements.
• Verification of Vector Function: Successfully validated that TAL and C3H genes can enhance brightness through transient transformation of tobacco leaves. The GRF3-RtTAL and GRF3-AtC3H modules can respond to formaldehyde signals.
• Plant Synthetic Biology: Evaluated the impact of the plant growth cycle on the transient transformation efficiency of tobacco leaves and its applications in plant synthetic biology.
• Luminous Plant Sensors: Explored how luminous plants can be developed into biosensors and the feasibility of their interaction with humans.

We conducted computational analyses of C3H proteins to explore the relationship between their structure and function. Eight C3H sequences from six plant species were collected and analyzed for motif consensus, tertiary structure, and predicted active sites. The results indicated a high level of structural conservation across the motifs and tertiary structures of the C3H proteins (Figure 1 & Figure 2). Despite this overall structural similarity, differences in light intensity among the members were observed, leading to the hypothesis that slight structural variations at the active sites may affect catalytic activity.

The docking simulation results (Figure 3) highlighted key residues involved in binding the substrate, p-coumaric acid. Notably, residues such as R436 and G432 in At_CYP98A3 were conserved across homologs but displayed subtle differences in neighboring residues, which may contribute to variations in enzyme efficiency and bioluminescence. These findings provide valuable insights into how small structural differences at the active site can influence the overall catalytic performance of C3H proteins, laying the foundation for further research and optimization of these enzymes for enhanced bioluminescence.

In three rounds of DBTL (Design-Build-Test-Learn), we successfully constructed 13 expression vectors. Each recombinant vector contains basic regulatory sequences, CDS sequences, and fused reporter genes. The target gene sequences were amplified using PCR cloning and the successful amplification was confirmed by agarose gel electrophoresis, showing bands of expected sizes. Using seamless cloning (or homologous recombination), these sequences were then inserted into the desired positions of the main vector, followed by transformation into E. coli TOP10 cells. Single colonies were picked for shaking culture, colony PCR, and agarose gel electrophoresis verification. The correct single colony transformants were confirmed by DNA sequencing.

The correct single colony transformants were then subjected to shaking culture, plasmid extraction, and transformation into competent Agrobacterium GV3101 cells. Single colonies were picked for shaking culture, colony PCR, and agarose gel electrophoresis verification to confirm the correct Agrobacterium single colony transformants, which were then stored in glycerol stock (culture: 50% sterile glycerol = 1:1, mixed and stored at -80°C).

In this project, the vector backbone for the insertion of the target gene CDS is the modified pS1300-GFP, based on the pCAMBIA1300 backbone. The insertion site is located in the multiple cloning site (MCS) between the Super promoter and GFP, specifically between Hind III and Kpn I (indicated by the green arrow in Figure 2A).

Figure 3 shows the process of amplifying the target gene RtTAL CDS (BBa_K5218005). Initially, due to improper handling, the desired band was not observed in the electrophoresis (Figure 3A). We then performed PCR cloning again using the plasmid as a template (Figure 3B). However, due to sample spillage on the floor during gel loading, the recovered sample quantity was insufficient. We used the PCR product as a template for cloning again and obtained a new product (Figure 3C) with a length of 2206 bp. The re-amplified product resulted in non-specific bands, but we decided to proceed with direct purification and recovery. The double enzyme digestion of the plasmid GFP was performed on this sample (with extended digestion time followed by direct product purification). The recombinant single colony E. coli verification showed bands of different sizes. We selected the expected results for sequencing (Figure 3E), and the sequencing results confirmed successful construction.

Figure 4 shows the successful amplification of the target gene RcTAL CDS (BBa_K5218001), with a length of 1657 bp. The digested GFP product also appears higher than the circular control plasmid (as linearized plasmids migrate more slowly than circular plasmids during electrophoresis), indicating successful digestion (Figure 4A). The PCR validation of the recombinant monoclonal colonies confirms that most of the monoclonal colonies were successfully constructed, as verified by PCR and sequencing (Figures 4C and 4D).

Figure 5 shows the successful amplification of the target gene NoRtTAL CDS (BBa_K5218025), with a length of 2206 bp. The GFP digestion product is at the same position as the circular control plasmid, indicating that an extended digestion time is required before product purification (Figure 5A). PCR validation of the recombinant monoclonal colonies confirms that most of the monoclonal colonies were successfully constructed, as verified by PCR and sequencing (Figures 5C and 5D).

Figure 6 shows the successful amplification of the target gene AtC3H CDS (BBa_K5218002) through two rounds of PCR. The first round yielded a product with a length of 1525 bp, and the second round produced a product with a length of 1536 bp. Similarly, PpC3H CDS (BBa_K5218007) was also successfully amplified in two rounds, with the first PCR product measuring 1786 bp and the second 1601 bp (Figures 6A and 6B), resulting in products without stop codons and with recombinant overhangs.The PCR validation of monoclonal colonies for the recombinant pS1300-AtC3H-GFP confirmed that most of the colonies were correctly constructed and verified through PCR and sequencing (Figures 6C, 6F, and 6G). However, when constructing pS1300-PpC3H-GFP, colony PCR validation of the first connection plates showed no bands (Figure 6F, lanes 13-21), and multiple attempts to isolate colonies still resulted in no bands. Seamless cloning was repeated (Figure 6E), and subsequent colony PCR validation (Figure 6H) showed the expected bands. Sequencing was successful (Figure 6I).

Figure 7 shows the successful two-round PCR amplification of the target gene NtC3H CDS (BBa_K5218011). The first round of PCR produced a product with a length of 1782 bp, and the second round produced a product with a length of 1536 bp (Figure 7A and Figure 7B), ultimately obtaining a product without a termination codon and with recombinant overhangs. PCR validation of the recombinant single colonies confirmed that most of the single colonies were successfully constructed, as verified by PCR and sequencing (Figure 7D and Figure 7E).

Figure 8 shows the successful two-round PCR amplification of the target gene SlC3H CDS (BBa_K5218013). The first round of PCR produced a product with a length of 1573 bp, and the second round produced a product with a length of 1545 bp (Figure 8A and Figure 8B), ultimately obtaining a product without a termination codon and with recombinant overhangs. PCR validation of the recombinant single colonies confirmed that most of the single colonies were successfully constructed, as verified by PCR and sequencing (Figure 8D and Figure 8E).

Figure 9 shows the successful two-round PCR amplification of the target gene CrC3H CDS (BBa_K5218009). The first round of PCR produced a product with a length of 1688 bp, and the second round produced a product with a length of 1569 bp (Figure 9A and Figure 9B), ultimately obtaining a product without a termination codon and with recombinant overhangs. PCR validation of the recombinant single colonies confirmed that most of the single colonies were successfully constructed, as verified by PCR and sequencing (Figure 9D and Figure 9E).

The vector backbone used for the target gene promoter in this project is pS1300-GUS, an improved version based on the pCAMBIA1300 backbone. The insertion site replaces the Super promoter sequence between the BamH I sites on both ends (indicated by the blue arrow in Figure 10A).

Figure 11 shows the successful amplification of the target gene GRF3 promoter (BBa_K5218017), with a length of 1786 bp. The GUS digestion product appears slightly higher than the circular control plasmid, indicating successful digestion (Figure 11A). PCR validation and sequencing of the recombinant single colonies confirmed successful vector construction (Figure 11C and Figure 11D). During PCR validation of single colonies after transforming the recombinant plasmid into Agrobacterium GV3101, initial attempts failed to produce the expected bands due to undetected PCR machine malfunctions, leading to multiple primer changes. Ultimately, only three single colonies could be verified (Figure 11G, blue box).

Figure 12 shows the successful amplification of the target gene GS1 promoter (BBa_K5218019), with a length of 997 bp (Figure 12A). PCR validation and sequencing of the recombinant single colonies confirmed successful vector construction (Figure 12C and Figure 12D). After transforming the recombinant plasmid into Agrobacterium GV3101, single colony PCR validation was also successful (Figure 12F).

Figure 13 shows the successful amplification of the target gene MDH1 promoter (BBa_K5218021), with a length of 341 bp. The GUS digestion was incomplete, resulting in two bands, indicating the need to increase the digestion time before product recovery (Figure 13A). PCR validation and sequencing of the recombinant single colonies confirmed successful vector construction (Figure 13C and Figure 13D). After transforming the recombinant plasmid into Agrobacterium GV3101, single colony PCR validation was also successful (Figure 13F).

The Super promoter in pS1300-RtTAL-GFP and pS1300-AtC3H-GFP was replaced with the GRF3 promoter, still using BamH I for single enzyme digestion to remove the Super promoter sequence. Since we used a seamless cloning method, the PCR products required corresponding homologous arm overhangs for recombination. The 5' end of the GRF3 sequence was connected to the basic backbone of both vectors, in the same position as the GUS vector, so the homologous overhangs were the same. However, the 3' end was connected to the RtTAL and AtC3H sequences, respectively, so the recombination overhangs were specific to each gene. The figure shows the successful amplification of the GRF3 promoter (Figure 14A, lanes 5 and 6). After replacing the PCR machine, both E. coli and Agrobacterium single-colony PCR validations confirmed successful vector construction (Figure 14F-L).

Additionally, we attempted to amplify other C3H genes from different plant materials, such as RcC3H from rose and other homologous C3H sequences from the same species. However, due to material variability and growth cycle limitations, we did not obtain the expected bands. The vector construction process was also not very smooth, and we encountered multiple failures, including operational errors, equipment malfunctions, and unknown reasons. Fortunately, through repeated attempts, we became more proficient in various experimental methods and learned important considerations for repeating experiments. You can refer to this link for further details.

We successfully contacted Professor Du from the glowing plant research team at Zhejiang University, who was willing to provide us with the first-generation glowing vector FBP-22 (introducing FBP, including LUZ, H3H, CPH, and HispS genes). We collaborated with the Mammoth Education system to construct the transgenic tobacco line Nicotiana benthamiana FBP-22/FBP-T2. The initial transgenic self-luminous tobacco lines FBP-22/FBP-T2 were proven to be self-luminous. However, the intensity of the luminescence was limited and did not meet our practical application goals. Our team focuses on enhancing plant luminescence and exploring further applications, using the FBP-T2 tobacco line as a control and genetic engineering material for the functional verification of TAL and C3H in FBP.

We separately transformed Agrobacterium GV3101 strains with pS1300-GFP and pS1300-gene CDS-GFP constructs, followed by transient transformation of tobacco leaves through injection. After 48 hours, transgenic tobacco leaves were examined under a fluorescence microscope. The results showed GFP fluorescence signals from the gene CDS-GFP fusion proteins in the epidermal cells of tobacco leaves, indicating the successful engineering of each pS1300-gene CDS-GFP construct.

We observed the phenotypes in a completely dark setting using a DSLR camera (parameters: ISO 6400, aperture F2, shutter speed 30s). The results showed that the experimental groups transiently expressing the 8 pS1300-gene CDS-GFP constructs exhibited higher brightness compared to the Control (only injected vector pS1300-GFP) and Blank Control, aligning with our expectations. This preliminary result indicates that the successfully constructed TAL and C3H genes can enhance luminescence intensity.

To visually demonstrate that our constructs can enhance the self-luminescence brightness of the first-generation tobacco line FBP-T2, we performed uniform dotting on the injection areas and analyzed luminescence brightness using ImageJ software by measuring grayscale values. The results showed that these TAL and C3H genes effectively enhanced luminescence intensity. The C3H group consistently exhibited higher brightness than the Control (only injected vector), indicating that the C3H gene can enhance luminescence intensity. The highest brightness of AtC3H reached a grayscale value of 49, indicating that the AtC3H gene performed best in tobacco. Similarly, the TAL group enhanced luminescence intensity, with RtTAL achieving the highest brightness of 51 grayscale value, indicating that the RtTAL gene performed best in tobacco. Among the constructs, pS1300-RtTAL-GFP showed the highest brightness peak after injection, followed by pS1300-AtC3H-GFP, further validating the phenotypic results.

In addition to data comparison and analysis, we also analyzed the variability of the data. As shown in the figure below, in the C3H group, AtC3H had a relatively higher peak and shorter length, with a more concentrated distribution, indicating the most significant growth in AtC3H. In the TAL group, RtTAL also showed a relatively higher peak and shorter length with a dense distribution, demonstrating that RtTAL had the most growth. This confirms that the data reflected in the previous figure are generally accurate, and that AtC3H in the C3H group and RtTAL in the TAL group are more densely clustered, indicating more stable transformation repeatability.

Additionally, as glowing plant pets, our focus is on whether they can respond as switches to emotional gases secreted by humans in the environment. Due to limitations in Human Subjects Research Policy for ethical safety and the difficulty of human-related experiments, we chose formaldehyde, a common harmful gas in the environment, as a controllable variable parameter in our experiments.

We conducted transient infection and transformation of tobacco leaves using the blank control (0.5x PBS), positive control (pS1300-GUS), and experimental group (p1300-promoter-GUS constructs). After 12 hours of injection, the H2O control and 2 mM HCHO treatment were sprayed under the same conditions and cultured in a sealed environment for 36 hours. Samples were taken, and GUS staining and decolorization were performed at 48 hours.

Results showed that in the blank control group (0.5x PBS), neither H2O nor formaldehyde spraying induced any GUS signal. In the positive control group (pS1300-GUS), both H2O and formaldehyde treatments resulted in deep blue GUS signals, with formaldehyde treatment showing an even deeper blue. In the experimental group (p1300-MDH1-GUS), GUS signals were observed with both H2O and formaldehyde, but the blue color was weaker after formaldehyde treatment, and GUS signals were weaker than those in the GUS control group, indicating that formaldehyde treatment inhibited MDH1 promoter expression, though not significantly. In the experimental group (p1300-GS1-GUS), blue signals appeared after H2O treatment, but almost no GUS expression was observed after formaldehyde treatment, suggesting that GS1 promoter expression was inhibited by formaldehyde. In the experimental group (p1300-GRF3-GUS), blue signals were visible after H2O treatment, but no GUS expression occurred after formaldehyde treatment, showing that GRF3 promoter expression was significantly inhibited by formaldehyde.

Overall, the promoter-GUS modules showed significant phenotypic responses to formaldehyde. After formaldehyde treatment, the promoters' expressions were inhibited to varying degrees. The p1300-GRF3-GUS treatment exhibited the most notable difference, with the GRF3 promoter completely inhibiting GUS gene expression in response to formaldehyde, resulting in no blue-stained tissues in the transformed leaves.

Next, we used the GRF3 promoter, which showed the most significant phenotypic differences, to replace the Super promoter sequence in the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP vectors, creating constructs p1300-pGRF3-RtTAL-GFP (BBa_K5218023) and p1300-pGRF3-AtC3H-GFP (BBa_K5218024). This combined the formaldehyde-responsive regulation module with luminescence-enhancing genes to develop formaldehyde-responsive glowing plants.

The results after 36 hours of treatment with 2 mM HCHO showed that the control group (pS1300-GFP) expressed normal luminescence under water treatment, but luminescence was significantly suppressed under formaldehyde treatment, becoming very faint. In the experimental group p1300-pGRF3-RtTAL-GFP, where the Super promoter was replaced with the GRF3 promoter, normal luminescence was observed after water treatment, with brightness significantly higher than that of the GFP control group, and the brightness was almost unaffected by formaldehyde treatment. In the pS1300-RtTAL-GFP control group, the brightness did not change significantly after either water or formaldehyde treatment, though luminescence slightly weakened under formaldehyde, and overall brightness was higher than in the GFP group. This indicates that the GRF3 promoter responds to formaldehyde to some extent, but the luminescence difference is small, making it difficult to draw further conclusions.

In the formaldehyde response experiment of the p1300-pGRF3-RtTAL-GFP vector, the tobacco seedlings used were at an incorrect growth stage, making the injection of the infection solution difficult and resulting in irregular luminescent areas and significant leaf damage. This greatly affected the brightness analysis and visual perception of the results. Therefore, the results are for reference only, and after reflecting on the reasons, we conducted a more comprehensive second functional verification.

We observed a similar pattern in the p1300-pGRF3-AtC3H-GFP experiments as seen in the pGRF3-RtTAL module. To better contrast this phenomenon, we adopted a different photography method. As shown in the figure below, the pS1300-GFP control group was significantly inhibited by formaldehyde, resulting in extremely faint luminescence. In the p1300-pGRF3-AtC3H-GFP experimental group, luminescence slightly increased after formaldehyde treatment compared to water treatment. The brightness of the pS1300-AtC3H-GFP control group was generally consistent, with formaldehyde treatment resulting in slightly lower brightness than the p1300-pGRF3-AtC3H-GFP group.

Overall, the GRF3 promoter can respond to formaldehyde, enhancing the luminescence of glowing plants, though the effect is not very pronounced. Additionally, the AtC3H gene allows plants to maintain near-normal luminescence under formaldehyde inhibition, demonstrating the gene's stability. The results of this experiment were improved based on verification from different batches, using more mature tobacco plants and directly photographing the luminescent areas of the injected leaves for clearer results.

Excitingly, the results from all the above functional validation experiments demonstrate that both TAL and C3H genes exhibit high luminescence stability. From the transient transformation verification of the eight pS1300-gene CDS-GFP constructs in tobacco leaves, we confirmed that the RtTAL and AtC3H genes effectively enhance the self-luminescence brightness of the first-generation transgenic tobacco, with grayscale values more than doubling compared to the control. The GRF3 promoter effectively responded to formaldehyde, meeting our expectations and successfully creating formaldehyde-responsive glowing plants. This experiment provides a promising validation of the feasibility of detecting gases released by human emotional fluctuations in the future.

Since the formaldehyde used in the experiments can cause some harm to plants, it is important to note that the overall physiological activity of synthetic biology within plants is inherently complex. GUS staining is commonly used to study gene promoter expression in plants, and we attempted to apply it to explore the effects of environmental factors on gene expression. The experiential analysis of the results can be referenced here.

Synthetic biology, simply put, starts with the most basic elements of life, step by step constructing parts and components, and using the principles of modern engineering to synthesize artificial organisms with specific functions based on the principles of biological composition. It aims to create artificial biological systems that can function like circuits. Synthetic biology applies engineering concepts, using existing biological components or modules to design and construct biological systems with specific biological functions. The luminescent systems found in glowing organisms, especially the biological components related to light-emitting reactions, provide valuable materials for the modification of organisms. These components from luminous organisms are used as biosensors or reporter systems for monitoring the environment or scientific research, or for constructing glowing plants as plant pets—an idea we are exploring.

Glowing plants represent a fascinating area within synthetic biology, combining knowledge from biology, genetic engineering, and botany to create organisms with new functions. By using synthetic biology approaches, researchers can introduce foreign genes into plants, enabling them to express luminescent proteins and thus granting the plants the ability to glow.

1. Luminescence Mechanism: In synthetic biology, researchers typically utilize luminescent genes extracted from other organisms, such as fireflies, bioluminescent mushrooms, or marine organisms. These genes encode proteins that produce luminescence, such as luciferase or similar substances. Through genetic engineering techniques, these genes can be introduced into a plant’s genome, allowing the plant to glow under specific conditions.

2. Applications and Significance: Glowing plants are not just scientific achievements; they also hold potential for practical applications. For example, glowing plants can act as environmental sensors, detecting heavy metals, pollutants, or pathogenic microorganisms in the soil, or even harmful gases. Additionally, they can be used for landscape beautification or as green lighting solutions.

3. Challenges and Ethical Considerations: Although glowing plants present exciting scientific and practical prospects, their development and use also face challenges. Ensuring that gene insertion does not affect the plant's normal growth or disrupt the ecosystem is a critical consideration. Moreover, ethical issues and public acceptance must also be carefully addressed. In summary, glowing plants represent an intriguing branch of synthetic biology that showcases the potential of biological engineering. They not only expand our understanding of plants but also offer new possibilities and challenges for the future of biotechnology.

Plant Growth Cycle Records:

During the seedling cultivation period, we observed the following: 1. As shown in the raw data in the Excel sheet below, many leaves were still in the budding stage in the early period, so no growth records were taken; only significant growth was recorded later. 2. In the later growth records, some bottom leaves exhibited slowed growth, fluctuations in leaf length, and even wrinkling, which could be due to mild dehydration or the plant sacrificing nutrients from the lower leaves to support the growth of the upper leaves. 3. After more than four days of recording, the growth slope of newly emerged leaves was significantly steeper than that of the older leaves, indicating that the new leaves grew much faster than the older ones. This may have a beneficial effect on plant growth (apical dominance).

Due to the growth of the leaves, the gravity of the leaves will lower the height of the plant, and the contact with the plant during measurement may change the height of the plant, so the plant height here is unstable and there is a large fluctuation.

According to the chart, the plant height was generally slower in the early stage. The first and second batches both had large fluctuations in plant height between August 8 and August 14. Because the first batch and the second batch were not planted at the same time, the tobacco should be affected by environmental factors, and therefore, the instability of plant height makes plant height not a standard to measure the development of tobacco.

The Impact of Seedling Stage on Experimental Results:

In plant experiments, we typically use Agrobacterium-mediated transient transformation of tobacco leaves for rapid functional validation. When infecting leaves, older leaves are generally not selected under non-specific conditions for the following reasons:

1. Cell Activity: Older leaves have lower cell activity and reduced metabolic activity, which are not conducive to the expression of foreign genes and reduce transformation efficiency.

2. Tissue Structure: Older leaves have thicker cell walls and a harder texture, which may hinder the transfer and absorption of foreign DNA, reducing transformation efficiency.

3. Health Condition: Older leaves may harbor more pathogens or be more affected by environmental stress, making them less healthy than younger leaves, which could impact the accuracy of experimental results.

4. Physiological State: Younger leaves are in a physiological state more suitable for gene expression and protein synthesis, enhancing the effectiveness of transient expression.

Therefore, selecting younger leaves for transient transformation experiments in tobacco can improve transformation efficiency and gene expression levels, ensuring the reliability and accuracy of the experimental results.

To determine the optimal injection period for younger leaves, our data analysis indicated that around the third week (approximately 16 days) after transplanting is suitable for preparing infection solutions and conducting bacterial culture shaking. We then performed transient injections at various growth stages of the tobacco seedlings, finding notable differences between early-stage new leaves and mature leaves (26 days after germination) at the injection stage.

Compared to younger leaves, mature tobacco leaves are fully expanded, have smoother surfaces, and clearly defined veins with fewer small veins, allowing a single injection to spread the infection solution over a larger area, often covering more than one-third of the leaf area, thereby reducing the number of injections needed. In contrast, immature leaves are not fully smooth, sometimes exhibiting uneven surfaces and numerous fine veins, making injection more challenging and resulting in a smaller spread area per injection, often not exceeding a 2 cm diameter circle. This necessitates multiple injections. Under these conditions, using overly young leaves for injection, such as when comparing luminescence differences, causes significant damage to the leaves and greatly affects the accuracy and stability of functional validation results.

Therefore, it is advisable to schedule bacterial culture shaking and infection solution preparation around the third week (approximately 16 days) after transplanting, with infection and phenotypic observation conducted one month from the initial sterilization treatment.

Our project aims to create glowing plant pets that heal the human spirit through luminous plants. By enhancing plant luminescence intensity based on fungal bioluminescence pathways, we achieve visibility at night. We design biosynthetic pathways within plants to significantly increase the production of caffeic acid, thereby enhancing overall bioluminescence. Beyond this, we combine dark therapy on a mental health level with glowing plants to soothe the human mind and alleviate stress.

Use of Plant Chassis (Glowing Plants):

The plant chassis offers unique advantages over microbial chassis in terms of cell structure, product affinity, safety, and storage. The development of plant tissue culture systems, such as hairy root cultures, has accelerated the commercialization and industrialization of synthetic biology in plant-derived natural products.

Cell Structure: Plants have a finely compartmentalized cellular structure that supports the proper functioning of membrane-localized exogenous enzymes, particularly cytochrome P450 (CYP450) enzymes.

Species Compatibility: Plant chassis are better suited than microbial chassis for expressing exogenous genes from plant species. Natural product synthesis in plants often involves multiple biochemical reactions and active enzymes, some of which are absent in microbial chassis, requiring additional genes and hindering the achievement of target products.

Post-Translational Modification: Plant chassis possess comprehensive post-translational modification systems that can structurally modify natural products, ensuring the functionality of heterologous proteins.

Cost Reduction: By synthesizing and accumulating natural products within the plant chassis, costs associated with cultivation, transportation, and preservation can be significantly reduced.

Stable Gene Integration: Plant chassis can integrate exogenous genes into the host genome more stably, allowing these genes to be inherited by the next generation. In contrast, prokaryotic chassis often rely on recombinant plasmids that exist independently of the host genome and can be lost during culture, leading to the loss of exogenous genes.

Bioluminescence Mechanism in Synthetic Biology:

Bioluminescence is a widespread natural phenomenon observed in approximately 10,000 species. It refers to the phenomenon where living organisms emit light, or extracts of these organisms emit light in the laboratory. This luminescence does not rely on the absorption of light by organisms but is a special type of chemiluminescence with nearly 100% efficiency in converting chemical energy into light energy. It is also a form of oxidative luminescence. The general mechanism of bioluminescence involves chemical substances synthesized by cells that, under the action of a special enzyme, convert chemical energy into light energy.

Recently, a bioluminescence pathway using caffeic acid as a substrate was discovered in fungi, producing green fluorescence at a wavelength of approximately 520 nm. This pathway includes four key steps:

1. HispS Enzyme: Uses ATP to convert caffeic acid into hispidin.

2. H3H Enzyme: Hydroxylates hispidin to form 3-hydroxyhispidin (fungal luciferin).

3. Luciferase: Fungal luciferin is oxidized by oxygen, producing an excited high-energy intermediate, emitting green fluorescence at 520 nm, along with carbon dioxide and caffeoylpyruvate.

4. CPH Enzyme: Hydrolyzes caffeoylpyruvate into pyruvate and caffeic acid, re-entering the cycle [1].

Rapid Screening and Validation of Function Based on Luminescence Signal:

The main objective of this study is to screen genes related to caffeic acid biosynthesis and introduce them into tobacco, using genetic engineering to create a new type of bioluminescent plant. We expect these experiments to significantly enhance the efficiency of caffeic acid synthesis, thereby strengthening the bioluminescent characteristics of plants. Based on the principles of synthetic biology and plant synthetic biology, we optimized the caffeic acid biosynthesis pathway by replacing the key catalytic enzymes RtTAL and AtC3H. Through transient injection at the optimal stage of plant growth, we successfully validated that these modifications can enhance plant bioluminescence. Additionally, based on literature, we identified the GRF3 gene, which regulates plant responses to formaldehyde, and utilized its promoter to modify key catalytic enzyme expression modules. By integrating this into the caffeic acid synthesis pathway, the system activates key catalytic enzymes when formaldehyde levels are high in the environment, leading to increased caffeic acid synthesis and enhanced luminescence. This work aims to create visually detectable bioluminescent plants that serve not only as comforting “pets” but also as innovative biological indicators for environmental monitoring, particularly for detecting harmful gases such as formaldehyde.

Based on the principles of plant synthetic biology, we observed the plant growth cycle, expanded the use of Agrobacterium-mediated transient transformation of tobacco leaves, and studied how plants respond to formaldehyde, utilizing this response to enhance luminescence intensity.

Studies have reported that the expression of the At14-3-3PSI (At14PSI/GRF3) gene in Arabidopsis is rapidly upregulated under formaldehyde stress. The knockout or reduction of AtGRF3, AtMDH1, or AtGS1 genes lowers the plant's ability to absorb and resist HCHO. However, overexpression of AtGS1 and AtMDH1 genes in the At14-3-3 psi mutant restored Arabidopsis's uptake and resistance to HCHO. This indicates that AtGS1 and AtMDH1 are indeed indispensable for the metabolism of HCHO in Arabidopsis [2].

We designed primers to clone the promoter sequence of the AtGRF3 gene and replaced the promoters of key catalytic enzyme genes in the caffeic acid synthesis pathway. When formaldehyde concentration in the plant increases, the promoter responds and regulates caffeic acid synthesis, increasing substrate levels and enhancing luminescence intensity.

Our luminescent plants that respond to formaldehyde can be considered plant-based biosensors. Plant-based biosensors have significant advantages in detecting soil and water pollution, as they can absorb chemicals from the surrounding environment as they grow naturally and exhibit morphological changes. This makes them more convenient and efficient than existing electronic detection devices and more responsive to low-concentration pollutants. Additionally, plant biosensors have the added benefit of not affecting the natural environment while detecting pollutants and metabolizing harmful substances.

Once the appropriate indicators are determined, ordinary people can visually observe their shape and quickly detect pollutants. In the future, we can continue to use the growth cycle data of Nicotiana benthamiana for transient functional validation of other genes to develop more application scenarios.

This innovative bioluminescent plant, as an ornamental plant, can also serve as a "pet" that soothes the soul. By increasing the concentration of caffeic acid, we aim for the bioluminescence to be visible to the naked eye and felt by the heart. By integrating our glowing plants into dark therapy, a method used in psychological treatment, we hope to relieve people's stress, release emotions, ease tension, and even help alleviate depression. Through this research, our goal is to promote the practical application of biotechnology in everyday life.

In the rapidly developing field of synthetic biology, there is an urgent need for robust bioethical frameworks to address emerging challenges. Due to its rapid growth, the ethical guidelines related to synthetic biology are not always comprehensive. This reality highlights the necessity for individuals working in this field to take responsibility for ensuring their progress adheres to principles of ethics, responsibility, and genuine benefit to the world.

Synthetic biology possesses extraordinary capabilities, enabling highly complex and modular systems to address some of humanity's most pressing issues through the standardization of biological components. For example, in our project, using plant synthetic biology, we successfully validated bioluminescent plants capable of detecting formaldehyde without harmful chemical reagents, thereby reducing environmental pollution and aligning with sustainable development principles.

However, the responsibility to assess the impact of our creations should not rest solely with governments and ethicists. While they are valuable resources, we, as individuals, have the duty to analyze our creations from a bioethical perspective and uphold the principles of scientific integrity.

[1] Mitiouchkina, T., Mishin, A.S., Somermeyer, L.G. et al. Plants with genetically encoded autoluminescence. Nat Biotechnol 38, 944–946 (2020).

[2] Xing Zhao, Xueting Yang, Yunfang Li, Hongjuan Nian, Kunzhi Li. (2023) 14-3-3 proteins regulate the HCHO stress response by interacting with AtMDH1 and AtGS1 in tobacco and Arabidopsis. Journal of Hazardous Materials, 458, 132036