Engineering

To achieve a higher and more controllable bioluminescence visible to the naked eye, we designed the project as three Design-Build-Test-Learn (DBTL) cycles. In the first cycle, we increased the concentration of substrates in the caffeic acid cycle using the Tyrosine Ammonia-Lyase (TAL) and p-Coumarate 3-Hydroxylase (C3H) genes, thereby enhancing the brightness of the plant’s bioluminescence, similar to adding more fuel to an engine to make it run faster. In the second cycle, we tested a regulatory module for key catalytic enzymes upstream of the caffeic acid pathway to further increase production, implementing a β-glucuronidase (GUS) staining module to enable the plant to respond to formaldehyde. In the third cycle, we combined the formaldehyde-responsive regulatory module with the enhanced bioluminescence genes, creating a plant that can respond to formaldehyde with increased light emission, much like adding an accelerator pedal to our engine.

We have obtained the first-generation stable transgenic line FBP-T2 seeds (also known as FBP-22) of bioluminescent tobacco from a research institution. While this line produces visible bioluminescence, its brightness is still far from meeting practical everyday use requirements [1].

Therefore, in this cycle, to enhance the bioluminescence intensity of the FBP-T2/FBP-22 line, we planned to screen upstream pathway genes that increase the substrate concentration in the caffeic acid cycle. After reviewing the literature and mapping the metabolic pathways that could boost the substrate concentration in the caffeic acid cycle, we identified the TAL gene [2] and C3H gene [3]. We constructed overexpression plasmids for these genes and introduced them into Escherichia coli TOP10 for positive monoclonal identification and amplification. The target plasmids were then transferred into Agrobacterium tumefaciens strain GV3101, followed by positive PCR identification. Using Agrobacterium-mediated transient transformation, we infected tobacco leaves to verify functional expression.

The pS1300-GFP (pCAMBIA1300 backbone) vector framework was provided by Mammoth Education Co., Ltd. The vector was linearized by double digestion with Kpn I and Hind III, followed by purification to obtain the linear plasmid.

The sequences for RcTAL, RtTAL (codon optimized), and NoRtTAL (non-codon optimized) were synthesized by BGI LHG and PCR-cloned using the plasmid as a template to obtain PCR products with homologous arms. For AtC3H, CrC3H, NtC3H, PpC3H, RcC3H, and SlC3H, the templates used for the first round of PCR cloning (to obtain PCR products with stop codons) were the cDNA from Arabidopsis thaliana (At), Catharanthus roseus (Cr), Nicotiana tabacum (Nt), Physcomitrium sphaericum (Pp), Rosa spp (Rc), and Solanum lycopersicum (Sl), respectively. A second round of PCR cloning was performed using these PCR products as templates to obtain homologous arm-containing PCR products.

Each gene fragment (purified PCR product with homologous arms) was ligated into the linearized vector using seamless cloning (In-Fusion Cloning) techniques. This produced the final verified plasmids, including:

• pS1300-RtTAL-GFP (BBa_K5218006)
• pS1300-AtC3H-GFP (BBa_K5218003)
• pS1300-RcTAL-GFP (BBa_K5218004)
• pS1300-NoRtTAL-GFP (BBa_K5218026)
• pS1300-CrC3H-GFP (BBa_K5218010)
• pS1300-NtC3H-GFP (BBa_K5218012)
• pS1300-PpC3H-GFP (BBa_K5218008)
• pS1300-SlC3H-GFP (BBa_K5218014)

The plasmid maps for each construct are shown below:

We successfully cloned and constructed eight vectors using different methods and transformed them into Agrobacterium tumefaciens GV3101, followed by positive clone verification via PCR, which was successful. Subsequently, we performed functional validation in the FBP-T2/FBP-22 tobacco lines using the transient transformation method. To observe the phenotypes, we photographed the plants in complete darkness with a DSLR camera set to ISO 6400, aperture F2, and a 30-second exposure. The bioluminescence brightness was analyzed using ImageJ to measure grayscale values.

As shown in Figures 1T-1 and 1T-2, the experimental data indicate that all eight experimental groups, which were transiently injected with pS1300-gene-GFP constructs, exhibited varying degrees of higher brightness compared to the control group (injected with only the pS1300-GFP vector). These preliminary results suggest that the successfully constructed TAL and C3H genes can effectively enhance bioluminescence intensity. Among them, the pS1300-RtTAL-GFP injection produced the highest brightness, followed by the pS1300-AtC3H-GFP injection. The phenotypic observation results further confirmed this finding (Figures 1T-3 and 1T-4).

Through the transient expression of the eight genes, we concluded that the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP constructs we designed and built can effectively enhance bioluminescence intensity. The phenotypic imaging results showed significantly higher brightness compared to the Control (only injected vector pS1300-GFP) and Blank Control, achieving brighter luminescence than the first-generation lines FBP-T2/FBP-22. These genes play a crucial role in increasing substrate concentrations in the caffeic acid cycle and boosting bioluminescence.

Additionally, we conducted multiple tests to extract RNA and obtain cDNA samples from various materials, stages, and parts of ornamental roses, but unfortunately, we were unable to clone RcC3H. During RNA extraction, we observed significant browning of the RNA precipitate, likely due to the high content of secondary metabolites leading to excessive impurities. Attempts to change RNA extraction methods were unsuccessful in reducing the impurity content. Due to time constraints and the availability of sample types, we decided to prioritize more critical tasks and abandon further attempts to obtain the RcC3H gene.

In conclusion, we have selected the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP vectors for subsequent experimental validation.

We aim for the plant to adjust its bioluminescence in response to environmental signals, showcasing the potential of synthetic biology in environmental sensing and response. After enhancing the bioluminescence, we wondered if the plant could detect human emotional fluctuations by sensing gases released through breathing or secretion. However, upon reviewing relevant studies, we found that collecting such gases for experiments is difficult and restricted by Human Subjects Research Policy.

As a result, we chose formaldehyde, a common and controllable environmental hazardous gas, as the ideal signal for monitoring. Through literature review and the NCBI database, we identified the promoter sequences for formaldehyde-responsive factors in plants, AtGRF3, AtGS1, and AtMDH1 [4]. We then constructed promoter-GUS vectors and used Agrobacterium-mediated transient transformation to verify GUS expression in tobacco leaves.

In this process, the pS1300-GUS (pCAMBIA1300 backbone) vector was digested with BamH I to remove the original strong Super promoter, resulting in a linearized vector, p1300-GUS.

The GRF3, GS1, and MDH1 promoters were PCR-cloned using Col-0 Arabidopsis DNA as a template, with homologous arms at both ends matching the BamH I sites of the GUS vector. These promoters were seamlessly cloned into the linear p1300-GUS vector. The constructs were then transformed into Escherichia coli TOP10 for sequencing verification, followed by plasmid extraction and transformation into Agrobacterium tumefaciens GV3101. The resulting Agrobacterium vectors—p1300-GRF3-GUS (BBa_K5218018), p1300-GS1-GUS (BBa_K5218020), and p1300-MDH1-GUS (BBa_K5218022)—were confirmed via PCR verification. The maps are shown below:

We performed transient transformation of tobacco leaves with the blank control (0.5x PBS), positive Agrobacterium control (pS1300-GUS), and experimental groups (p1300-GRF3-GUS, p1300-GS1-GUS, p1300-MDH1-GUS). After 12 hours of injection, we initiated the treatments under identical conditions by applying H2O as the control and 2mM formaldehyde spray. The plants were incubated in sealed conditions for 36 hours, with the spray treatment applied every 12 hours. After 48 hours post-injection, we collected samples and performed GUS staining and decolorization.

As shown in Figure 2T-2, in the blank control group (0.5x PBS), no GUS signal was detected after spraying with either H2O or formaldehyde. In the positive control group (pS1300-GUS), a strong blue GUS signal was observed after both H2O and formaldehyde treatments, with an even deeper blue coloration following formaldehyde application, indicating increased GUS expression.

In the experimental group treated with p1300-MDH1-GUS, GUS signals were observed after both H2O and formaldehyde treatments, but the blue coloration was weaker following formaldehyde treatment, and the GUS signal was generally weaker compared to the GUS control group. This suggests that the expression of the MDH1 promoter is inhibited by formaldehyde, though the level of inhibition is not very strong.

In the experimental group treated with p1300-GS1-GUS, blue coloration appeared after H2O treatment, but GUS expression was almost completely absent after formaldehyde treatment, indicating that the GS1 promoter is strongly inhibited by formaldehyde.

Similarly, in the experimental group treated with p1300-GRF3-GUS, blue coloration was observed after H2O treatment, but no GUS expression was detected after formaldehyde treatment, demonstrating that the GRF3 promoter is also inhibited by formaldehyde.

The results above indicate that the experimental groups with p1300-promoter-GUS showed significant phenotypic differences. After formaldehyde treatment, the expression of the promoters was inhibited to varying degrees. The p1300-GRF3-GUS group displayed the most pronounced differentiation, with the GRF3 promoter completely inhibiting the expression of the GUS gene in response to formaldehyde treatment, resulting in no blue-stained tissue in the transformed leaves. This outcome aligns with our concept of a formaldehyde-responsive bioluminescence monitor control element.

Next, we decided to replace the Super promoter on the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP vectors with the formaldehyde-responsive AtGRF3 promoter. This will combine the formaldehyde-responsive regulatory module with the bioluminescence enhancement genes, creating a plant that can respond to formaldehyde by adjusting its bioluminescence. This experiment aims to validate our hypothesis of whether the plant can detect gases released through human emotional fluctuations, such as those emitted through breathing or secretion.

In the first round of constructing the TAL or C3H gene fusions, we had already added a BamH I restriction site to the F-end adapter of the primers. Therefore, when removing the Super promoter from the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP vectors, we continued to use BamH I digestion to obtain linearized plasmids.

The R-end homologous arm sequence of GRF3 was modified through PCR cloning to match the corresponding homologous arms for RtTAL and AtC3H. Using seamless cloning, we constructed the new vectors p1300-pGRF3-RtTAL-GFP (BBa_K5218023) and p1300-pGRF3-AtC3H-GFP (BBa_K5218024). The plasmids, once confirmed by sequencing, were transformed into Agrobacterium tumefaciens GV3101 for positive PCR verification. The vector maps are shown below:

We continued to use Agrobacterium-mediated transient transformation of tobacco leaves for validation. After 12 hours of injection, the leaves were treated with H2O and 2mM formaldehyde, with formaldehyde treatment being refreshed every 12 hours. The plants were incubated in a sealed environment for 36 hours, and observations were made 48 hours post-injection.

The results showed that in the control group (pS1300-GFP), the leaves exhibited normal bioluminescence under water treatment, but the bioluminescence intensity was significantly suppressed after formaldehyde treatment, becoming extremely faint (Figure 3T-1). In the experimental group p1300-pGRF3-RtTAL-GFP, after replacing the promoter with GRF3, the leaves displayed normal bioluminescence under water treatment, with intensity noticeably higher than that of the control GFP group, and the bioluminescence intensity remained largely unaffected by formaldehyde treatment. In contrast, the pS1300-RtTAL-GFP control group showed little change in bioluminescence intensity after both water and formaldehyde treatments, with only a slight reduction in brightness following formaldehyde treatment, though still higher than the GFP control group.

The results suggest that the GRF3 gene does have some response to formaldehyde, but the difference in bioluminescence intensity is minimal, making it difficult to draw further conclusions.

In this experiment using the p1300-pGRF3-RtTAL-GFP vector in response to formaldehyde, the tobacco seedlings used were not at the correct stage for this experiment. The infiltration solution was difficult to inject, resulting in irregular bioluminescent areas and significant leaf stress, which affected both the brightness analysis and visual assessment. Therefore, these results are for reference only. After reflecting on the issues, we conducted a more complete second functional validation.

In the p1300-pGRF3-AtC3H-GFP experiment, we observed similar patterns to those found with the pGRF3-RtTAL module. To better illustrate this phenomenon, we used an alternative photography method. As shown in Figure 3T-2, the pS1300-GFP control group was significantly suppressed by formaldehyde, with bioluminescence becoming extremely faint. In the p1300-pGRF3-AtC3H-GFP experimental group, the bioluminescence slightly increased after formaldehyde treatment compared to water treatment. In the pS1300-AtC3H-GFP control group, the brightness remained consistent, with the formaldehyde-treated group showing slightly lower brightness compared to the p1300-pGRF3-AtC3H-GFP group.

The GRF3 promoter can respond to formaldehyde and increase the brightness of bioluminescent plants, though the effect is not very pronounced. Additionally, the AtC3H gene allows the plant to maintain near-normal luminescence under formaldehyde inhibition, demonstrating its stability. The results of this experiment were improved by verification across different batches, using tobacco plants grown for a longer period, and directly capturing the bioluminescence of the leaves after injection for clearer visualization.

Based on the results of these experiments, we can confirm that the GRF3 promoter does indeed respond to formaldehyde, and both the TAL and C3H genes exhibit high bioluminescence stability. This aligns with our expectations, and we successfully created a bioluminescent plant responsive to formaldehyde. This experiment also validates the feasibility of detecting gas signals released by human emotional fluctuations, such as breathing or secretion, in future studies.

However, the formaldehyde used in the experiment caused some damage to the plants, which affected the results. The Super promoter-GUS expression module did not perform as expected, which can be explained by the complexity of overall physiological activity within plants when applying synthetic biology. GUS staining is commonly used for studying promoter expression in plant genes, and our attempt to use it for studying environmental effects on gene expression highlights how it can be influenced by multiple factors, including plant growth and development. These findings provide useful reference points for other teams conducting related experiments. Below, Figure 3L-1 shows some explanations we received from online users when seeking help for these issues.

In the future, we aim to optimize the expression units of RtTAL and AtC3H by constructing them into a single vector for stable genetic transformation, with the goal of enhancing the brightness of the FBP-T2 line. We will design more refined experimental plans to monitor responses to environmental factors such as formaldehyde or emotional gases in stable plant lines. Additionally, optimizing protein expression and developing male sterility lines will also be important directions for future research.

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[2] Liu, Langqing, Liu, Hong, Zhang Wei, Yao, Mingdong, Li, Bingzhi, Liu, Duo, Yuan Yingjin. (2019) Engineering the biosynthesis of caffeic acid in saccharomyces cerevisiae with heterologous enzyme combinations. Engineering, 5(2), 9

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

[4] 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