Experiments

Between June 30, 2024, and August 26, 2024, our wet lab team successfully assembled 13 expression vectors and created new composite expression elements. Each recombinant vector included basic regulatory sequences, CDS sequences, and fused reporter genes. Using Agrobacterium-mediated transient transformation of tobacco leaves, we successfully validated that the TAL and C3H genes can enhance luminescence, while the promoters GRF3, GS1, and MDH1 can regulate the response to formaldehyde. Additionally, the constructed pGRF3-RtTAL and pGRF3-AtC3H modules also successfully responded to formaldehyde signals.

The following diagram presents the complete workflow and timeline of our experiment. This workflow clearly outlines the time schedule and procedural details for each experimental step, helping us to systematically advance the project. For more detailed experimental data and analysis results you can find here in Resutls section.

Experiments Workflow:

Experiments Timeline:

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).

The complete experimental SOP can be referenced here!

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).

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 3A).

We completed the construction of 8 vectors: pS1300-RtTAL-GFP (BBa_K5218006), pS1300-RcTAL-GFP (BBa_K5218004), pS1300-NoRtTAL-GFP (BBa_K5218026), pS1300-AtC3H-GFP (BBa_K5218003), pS1300-PpC3H-GFP (BBa_K5218008), pS1300-NtC3H-GFP (BBa_K5218012), pS1300-SlC3H-GFP (BBa_K5218014), and pS1300-CrC3H-GFP (BBa_K5218010). Additionally, we attempted to clone the C3H genes from other materials, but these were unsuccessful. We also successfully constructed pS1300-NtPKS-GFP, which was a deviation from the main project focus and was attempted without summarizing related data.

These constructs validated that the target genes TAL and C3H can enhance the self-luminescence intensity of the first-generation transgenic tobacco lines FBP-22/FBP-T2. The data shows that RtTAL and AtC3H achieved the highest increase in self-luminescence intensity.

Figure 1 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 1A). We then performed PCR cloning again using the plasmid as a template (Figure 1B). 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 1C) 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 1E), and the sequencing results confirmed successful construction.

Figure 2 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 2A). 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 2C and 2D).

Figure 3 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 3A). PCR validation of the recombinant monoclonal colonies confirms that most of the monoclonal colonies were successfully constructed, as verified by PCR and sequencing (Figures 3C and 3D).

Figure 4 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 4A and 4B), 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 4C, 4F, and 4G). However, when constructing pS1300-PpC3H-GFP, colony PCR validation of the first connection plates showed no bands (Figure 4F, lanes 13-21), and multiple attempts to isolate colonies still resulted in no bands. Seamless cloning was repeated (Figure 4E), and subsequent colony PCR validation (Figure 4H) showed the expected bands. Sequencing was successful (Figure 4I).

Figure 5 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 5A and Figure 5B), 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 5D and Figure 5E).

Figure 6 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 6A and Figure 6B), 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 6D and Figure 6E).

Figure 7 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 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).

Before August 4, 2024, we completed the transient transformation functional validation of the 8 gene CDS-GFP constructs.

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.

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.

We completed the construction of 3 vectors: (BBa_K5218018), (BBa_K5218020), and (BBa_K5218022). These constructs validated that the promoters GRF3, GS1, and MDH1 can respond to formaldehyde signals.

Figure 1 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 1A). PCR validation and sequencing of the recombinant single colonies confirmed successful vector construction (Figure 1C and Figure 1D). 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 1G, blue box).

Figure 2 shows the successful amplification of the target gene GS1 promoter (BBa_K5218019), with a length of 997 bp (Figure 2A). PCR validation and sequencing of the recombinant single colonies confirmed successful vector construction (Figure 2C and Figure 2D). After transforming the recombinant plasmid into Agrobacterium GV3101, single colony PCR validation was also successful (Figure 2F).

Figure 3 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 3A). PCR validation and sequencing of the recombinant single colonies confirmed successful vector construction (Figure 3C and Figure 3D). After transforming the recombinant plasmid into Agrobacterium GV3101, single colony PCR validation was also successful (Figure 3F).

Before August 18, 2024, we completed the transient transformation functional validation of the 3 promoter-GUS constructs.

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 5, 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.

We completed the construction of 2 vectors: (BBa_K5218023) and (BBa_K5218024). These constructs validated that the pGRF3-RtTAL and pGRF3-AtC3H modules can adjust luminescence intensity in response to formaldehyde signals.

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 1A, lanes 5 and 6). After replacing the PCR machine, both E. coli and Agrobacterium single-colony PCR validations confirmed successful vector construction (Figure 1F-L).

Before August 26, 2024, we completed the transient transformation functional validation of the 2 promoter-gene CDS constructs.

We then used the GRF3 promoter, which showed the most significant phenotypic differences, to replace the Super promoter sequence on the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP vectors, resulting in the constructs p1300-pGRF3-RtTAL-GFP (BBa_K5218023) and p1300-pGRF3-AtC3H-GFP (BBa_K5218024). This combined the formaldehyde-responsive regulatory module with luminescence-enhancing genes to create formaldehyde-responsive luminescent plants.

The results after treating with 2mM HCHO for 36 hours showed that the control group pS1300-GFP emitted normal luminescence under water treatment, but its luminescence was significantly suppressed and extremely weak under formaldehyde treatment. In the experimental group with the GRF3 promoter, p1300-pGRF3-RtTAL-GFP showed normal luminescence after water treatment, with brightness significantly higher than the GFP control group, and the brightness under formaldehyde treatment was almost unaffected. The pS1300-RtTAL-GFP control group showed little change in brightness under water and formaldehyde treatments, with a slight reduction in luminescence after formaldehyde treatment, although brightness was still higher than that of the GFP group. This suggests that the GRF3 gene has a certain response to formaldehyde, but the difference in luminescence intensity 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.

In summary, the GRF3 promoter can respond to formaldehyde and enhance the luminescence intensity of glowing plants, although the effect is not very pronounced. Additionally, the AtC3H gene enables the plant to maintain near-normal luminescence under formaldehyde suppression, demonstrating the stability of this gene. The results of this experiment were refined based on validations from different batches, using tobacco plants with a longer growth period and directly photographing the luminescence of leaves after injection to make the results more intuitive.