Overview ​

In this Engineering Cycle, the focus was placed on both the refinement of the pathway design for and the functional verification of the serotonin synthesis system.

Iteration 1 ​

Design ​

We initially designed the plasmid insert fragment containing p43-RBS-TPH(Flag)-terminator-p43-RBS-TDC(His)-terminator.

However, we have found that the tryptophan hydroxylase (TPH) enzyme requires the specific cofactor tetrahydrobiopterin/5,6,7,8-tetrahydromonapterin (BH4/MH4) (Figure 2) , while neither Escherichia coli nor Bacillus subtilis possesses the metabolic cycle pathway for MH4.

Build ​

Therefore, we modeled the reaction rate of the MH4 cycle using BH4 cycle enzymes (hPCDB1: Human Pterin-4α-carbinolamine dehydratase; hQDPR (/DHPR_HUMAN): Human quinoid dihydropteridine reductase) and MH4 cycle enzymes (PCD: Pterin-4α-carbinolamine dehydratase; DHMR: Dihydromonapterin reductase), respectively.

Test ​

According to the article by , we found that the substrate binding mode and the reaction mechanisms of pterin-4a-aminoalcohol dehydrogenase (PCD) isolated from Pseudomonas aeruginosa are basically the same as those of human PCD. That is to say, hPCBD1 can catalyze MH4 - 4a. Based on this finding, our model study focuses on the binding efficiency and catalytic efficiency of hQDPR (human Quinonoid Dihydropteridine Reductase) and DHMR toward MH2 (Figure 3).

Through model analysis, hQDPR exhibits higher catalytic efficiency toward MH2 than DHMR. We initially concluded that the two recycling enzymes of BH4 are applicable to the recycling of MH4; however, due to subtle differences in molecular structure, this recycling process is expected to generate new substances.

Subsequently, during our consultation with Professor Qi Feng, Vice Dean of the College of Life Sciences at Fujian Normal University, he indicated that the recycling enzymes of BH4 could be applied to MH4.

Learn ​

Therefore, we introduced two cycle enzymes, hPCDB1 and hQDPR, into the plasmid.

Meanwhile, to enable experiments in both Escherichia coli and Bacillus subtilis, we reselected the shuttle plasmid pWB980-ori, which is compatible with both bacterial strains.

Iteration 2 ​

Design ​

After obtaining the synthetic plasmids from the company, we first performed restriction enzyme digestion for verification.

Build ​

Plasmids of the control group (pWB980-ori) and the experimental group (pWB980-ori_m-hTPH1_hPCBD1_hQDPR-flag_TDC-his) were subjected to single restriction enzyme digestion (SacI-HF) and electrophoresis.

Test ​

Learn ​

Our target plasmid is confirmed to be correct, and we can utilize this plasmid for the next step of verification.

Iteration 3 ​

Design ​

To verify the expression of our target proteins, we detected the expression of proteins hQDPB-flag and TDC-his using Western blot.

Build ​

After we verified that the plasmid had been transformed into the bacteria, we cultivated the bacteria overnight and used the liquid to prepare sample for Western blot.

Test ​

Learn ​

We suspect that the incomplete transcription of hQDPR, which is the last gene, may be attributed to the fact that three genes are downstream of a single promoter.

Iteration 4 ​

Design ​

We aim to analyze whether the target gene is transcribed via RNA-based approaches such as reverse transcription-polymerase chain reaction (RT-PCR).

Build ​

First, we extracted RNA from both the control and experimental groups using an RNA Isolation Kit, followed by reverse transcription of the RNA into cDNA using the PrimeScript RT reagent Kit. Subsequently, we amplified the four sequences via touchdown PCR, and finally analyzed the results by electrophoresis.

Test ​

According to the results of reverse transcription-polymerase chain reaction (RT-PCR), all four target genes were successfully transcribed into full-length RNA.

Learn ​

The issue of unsuccessful gene transcription has been ruled out. To verify whether the protein with an abnormal molecular weight (size) detected by Western Blot (WB) corresponds to human quinoid dihydropteridine reductase (hQDPR), we may conduct product verification to determine the presence of functional hQDPR protein.

Iteration 5 ​

Design ​

All four of our target genes have been successfully transcribed into RNA; therefore, we intend to perform product detection.

Build ​

We generated a standard curve for the Serotonin ELISA Kit and subsequently detected the serotonin content in both the control group and the experimental group using this kit.

Test ​

By using the t-test, we can find that there is a significant difference between the experimental group and the control group. We conclude that pWB980-ori-TPH-hPCBD1-hQDPR-TDC can convert L-tryptophan into serotonin.

Learn ​

Based on the successful results of our RT-PCR, the successful TDC enzyme Western blot, and the positive outcomes of serotonin product detection, we can preliminarily conclude that the serotonin synthesis system is functional, even though the SNAT Western blot bands were incorrect for unknown reasons.

Overview ​

In this Engineering Cycle, the focus was placed on both the refinement of the pathway design for this system and the functional verification of the melatonin synthesis system.

Iteration 1 ​

Design ​

The synthesis of melatonin from serotonin requires the enzymes serotonin N-acetyltransferase (SNAT), and catechol-O-methyltransferase (COMT). Initially, we used the pWB980-ori vector backbone for constructing the expression plasmids of these two enzymes, just as we did for the serotonin synthesis plasmid.

Since it is necessary for us to introduce the dual plasmids into the bacteria, it is required to replace the high-copy pWB980-ori with the low-copy pHY300PLK, which has a different copy number characteristic.

Build ​

The plasmid pHY300PLK-SNAT-COMT was constructed using the T4 ligase method, which involved double enzyme digestion, electrophoresis, T4 ligation, transformation and sequencing verification.

Test ​

Learn ​

After we construct the plasmid, we can further detect the expression of the protein.

Iteration 2 ​

Design ​

To verify the expression of our target proteins, we detected the expression of proteins COMT-flag and SNAT-his using Western blot.

Build ​

After we verified that the plasmid had been transformed into the bacteria, we cultivated the bacteria overnight and used the liquid to prepare sample for Western blot.

Test ​

Learn ​

Since the protein size is incorrect, we are considering detecting whether it has been transcribed into RNA.

Iteration 3 ​

Design ​

We aim to analyze whether the target gene is transcribed via RNA-based approaches such as reverse transcription-polymerase chain reaction (RT-PCR).

Build ​

First, we extracted RNA from both the control and experimental groups using an RNA Isolation Kit, followed by reverse transcription of the RNA into cDNA using the PrimeScript RT reagent Kit. Subsequently, we amplified the four sequences via touchdown PCR, and finally analyzed the results by electrophoresis.

Test ​

According to the results of reverse transcription-polymerase chain reaction (RT-PCR), both target genes were successfully transcribed into full-length RNA.

Learn ​

The issue of unsuccessful gene transcription has been ruled out. To verify whether the protein with an abnormal molecular weight (size) detected by Western Blot (WB) corresponds to SNAT, we may conduct product verification to determine the presence of functional SNAT protein.

Iteration 4 ​

Design ​

Both target genes have been successfully transcribed into RNA; therefore, we intend to perform product detection.

Build ​

We generated a standard curve for the Melatonin ELISA Kit and subsequently detected the melatonin content in both the control group and the experimental group using this kit.

Test ​

Learn ​

After contacting the product technical consultant, we were informed that the suspected issue lies with the Sample Dilution they provided, which is mainly composed of 10% bovine serum, 0.2% Procline-300 preservative, and pH 7.0-7.4 PBS. We will conduct verification later. We suspect that there may also be interference from bacterial proteins.

Iteration 5 ​

Design ​

We need to re-test using the ELISA kit to identify the problematic reagent.

Build ​

We determined the Sample dilution, Amp medium, and pH=7.4 PBS buffer using the melatonin ELISA Kit. Then dilute the samples with PBS and perform the ELISA assay.

Test ​

Learn ​

The technician stated that this kit has not been tested using Escherichia coli (E. coli). Based on the data, it is likely that bacterial proteins are causing interference, and the affinity between the sample and the antibody may be insufficient.

Therefore, in our next experiment, we will reduce the interference of bacterial proteins by controlling the OD₆₀₀ of the bacterial suspension, and we will only use this kit for qualitative detection rather than quantitative detection.

Iteration 6 ​

Design ​

We will continue the melatonin ELISA experiments by controlling the bacterial concentration.

Build ​

To mitigate the influence of bacterial protein concentration, we regulated the optical density at 600 nm (OD₆₀₀), and on this basis, we continued to perform melatonin ELISA for both the control group and the experimental group.

Test ​

Learn ​

Unfortunately, due to constraints in time and technology, we were unable to obtain more precise results.

However, we can preliminarily speculate that pHY300PLK-SNAT-COMT is capable of converting serotonin into melatonin.

Overview ​

In this engineering cycle, our primary focus lies on the functional verification of the wax synthesis system.

Iteration 1 ​

Design ​

To realize our idea of coating the surface of lychees with wax, we reviewed literature, identified and constructed biological components, and ultimately designed our plasmid on SnapGene.

We designed a lactose-inducible biological logic circuit with a structure based on NOT gates, incorporating three proteins from the wax synthesis pathway. This enables bacteria to accumulate carbon sources in lactose-containing environments while synthesizing wax with accumulated fatty acid in lactose-free conditions. However, our design proved overly complex to conduct comprehensive experimental validation. After careful consideration, we determined that the wax synthesis pathway constitutes our core component. Consequently, we focused our attention on this pathway—specifically the three proteins WS/DGAT, AcrB, and G6PD—to validate their associated characteristics and properties.

Build ​

We constructed the plasmid according to the aforementioned design and obtained the physical plasmid. We also simultaneously ordered glycerol stocks containing this plasmid from a biosynthetic company for subsequent experimental validation.

Test ​

We designed a PCR assay to verify whether the relevant genes were correctly inserted into the vector. Our primary objective was to detect the three proteins required for wax synthesis.

We employed the Touch Down PCR method, setting up 10 gradient cooling amplification cycles followed by 25 conventional amplification cycles. Following PCR, we prepared a 1% agarose gel for electrophoresis, running the gel at 100V for 60 minutes. Finally, we visualized the bands using a blue light imager.

The results showed bands for G6PD and AcrB at their correct positions, with no bands appearing in the corresponding negative controls. This indicates that the sequences for these two genes on the vector are intact. However, no corresponding bands were observed for WS/DGAT.

Learn ​

Based on our experimental results, we conducted a retrospective analysis. By examining the primer sequences in SnapGene, we discovered that the primers used have significant nucleotide runs, leading to potential mispriming and increasing the likelihood of the primer forming secondary structures, such as hairpins, or self-dimers.

Iteration 2 ​

Design ​

Based on the previous experimental phase, we redesigned the primers to mitigate potential risks such as primer complementarity and insufficient primer specificity. We then reconfigured the PCR experiment, adjusting the annealing temperature to better align with the reaction conditions of the new primers.

Build ​

We conducted PCR experiments using both new and old primers. Primer set 1&2 are new, while primer set 3 is the previous one.

Test ​

PCR results indicate that our newly designed primer sets 1 and 2 successfully amplified the target band, confirming the successful design of the new primers. Meanwhile, primer set 3 and the negative control failed to amplify any bands, further validating the rigor of the experiment.

Learn ​

Based on the experimental results above, we have successfully verified the presence of sequences encoding three wax synthesis proteins—WS/DGAT, AcrB, and G6PD—on the plasmid. Following this finding, we will proceed to conduct more in-depth validation experiments.

Iteration 3 ​

Design ​

Based on the conclusions drawn from our aforementioned PCR validation experiments, we plan to proceed with the next step of protein validation experiments. We have designed a Western Blot assay to validate the protein we expressed.

Build ​

We performed electrophoresis using a 4-20% polyacrylamide gel. Given the molecular weights of our target proteins WS/DGAT, AcrB, and G6PD at 98.6 kDa, 101.4 kDa, and 57.4 kDa respectively, we designed and used nitrocellulose (NC) membranes with a pore size of 0.45 μm. Transfer conditions were uniformly set at 100 minutes and 300 mA. The WS/DGAT, AcrB, and G6PD proteins were tagged with flag, 6×His, and HA tags, respectively. Primary antibodies specific to these tags were used for incubation. Secondary antibodies corresponding to the species of the primary antibodies were then applied.

Test ​

The final membranes were immersed in ECL solution for several seconds, allowing the horseradish peroxidase covalently linked to the secondary antibody to oxidize luminol, producing luminescence. The results of our grayscale analysis using the t-test statistically rigorously validated our results.

However, when we placed it under the imaging system for observation, we found that only the WS/DGAT-flag produced the expected band at the anticipated position. Neither AcrB-his nor G6PD-HA produced bands at their expected positions.

Learn ​

Based on the WB experimental results, we have reflected on our experimental procedures. We speculate that the antibodies corresponding to AcrB-his and G6PD-HA may have been contaminated or inactivated, or that issues arose during experimental handling. We may need to repeat the experiments to obtain more accurate results, but this will be time-consuming.

Iteration 4 ​

Design ​

Due to the tight experimental schedule, after careful consideration, we had to abandon the subsequent Western Blot replication experiments and proceed directly to the target product detection phase. To detect wax content, we designed a series of experimental protocols, including absorbance measurements under conditions involving Oil Red O staining and oleic acid induction.

Build ​

We established five groups for full-wavelength absorbance detection. Since the study focused on the wax synthesis pathway initiated by acyl-CoA, oleic acid was added as the reaction substrate in all experimental groups. The groups were ultimately categorized as follows: PBS control group, PBS + Oil Red O control group, empty plasmid group, empty plasmid + oleic acid group, and target plasmid + oleic acid group. We employed normalization to ensure experimental results were unaffected by irrelevant factors such as bacterial culture concentration and background values.

Test ​

Oil Red O exhibits a strong absorption peak at 500 nm and demonstrates absorbance exceeding the control across the 400–650 nm range. Our experimental results corroborate this observation.

To eliminate extraneous factors and better analyze curve variations, we normalized the absorbance curves during analysis. Comparing the expression vector + oleic acid group with the other two control groups, we observed that the experimental group exhibited significantly higher absorbance at 500 nm than the other two groups. Additionally, it demonstrated higher absorbance below 650 nm, with the overall curve closely matching the absorbance curve of Oil Red O. However, due to experimental constraints, we did not replicate the experiment and thus could not perform a significant analysis.

Learn ​

Comparing the absorbance curves of the experimental group with the two control groups, wax formation in the experimental group is clearly discernible. This substantially confirms the success of our wax synthesis pathway design and plasmid construction, indicating preliminary realization of the intended function. We plan to conduct more in-depth validation in the next phase, such as testing its performance under different concentrations of oleic acid induction, verifying the activity of individual proteins, and exploring better improvement strategies. However, due to time constraints, we were unable to complete these planned steps.