During our experiment, we incorporated new components into the iGEM part and updated an existing part (Table 1). Specifically, we added metF (BBa_K5189000), pRSFDuet-1 (BBa_K5189005), pRSFDuet-metF-folA (BBa_K5189006), and pETDuet-ftfL-mtdA-fchA (BBa_K5189007). By constructing plasmids that drive the expression of these key enzymes, we aim to enhance the efficiency of the conversion process and thereby increase the production of L-5-MTHF in E. coli.

The pRSFDuet-metF-folA plasmid was constructed by selecting and amplifying the metF and folA genes to optimise the L-5-MTHF production pathway. The pRSFDuet-1 vector was chosen due to its capability to accommodate multiple gene insertions and its strong, regulated expression under the T7 promoter. The metF gene (894 bp) and folA gene (480 bp) were amplified via PCR and inserted into the pRSFDuet-1 vector.

Figure 1: The gel electrophoresis validation of metF(Left) and folA(Right) nucleic acids.

The metF and folA genes were successfully amplified using PCR, yielding bands of 894 bp and 480 bp, respectively. The metF gene was inserted into the pRSFDuet-1 vector by digestion with BamHI and HindIII, while the folA gene was inserted into the vector by digestion with NdeI and XhoI. The recombinant plasmid was then transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as evident from the expected band sizes in gel electrophoresis.

Upon verifying the successful amplification of the targeted plasmid, they were then transformed into E.coli DH5α. We selected colonies and sequenced them.

Figure 2: Transformation plate of pRSFduet-metF-folA(A);Enzyme digestion verification for DH5α: pRSF-metF (B),pRSF-metF-folA(C);Sequencing results of pRSFDuet-metF-folA (D).

The connected vectors were verified by enzyme digestion, and the exact target bands were obtained respectively. The first two lanes show successful enzyme digestion verification for pRSF-metF-folA, Which indicates that the destination segment is successfully connected.

The pETDuet-ftfL-mtdA-fchA plasmid was constructed with the objective of enhancing the L-5-MTHF synthesis pathway by co-expressing the ftfL, mtdA, and fchA genes. The pETduet-1 vector was selected for its dual-expression system, utilising the T7 promoter to drive synchronised expression of these critical enzymes. The ftfL gene (1685 bp) was inserted first, followed by the mtdA-fchA fragment (1488 bp), ensuring that all genes were under the control of the T7 promoter for optimal co-expression in E. coli BL21(DE3).

Figure 3: The gel electrophoresis validation of ftfL(Left), mtdA, and fchA(Right) nucleic acids.

The ftfL gene (1685 bp) and the mtdA-fchA fragment (1488 bp) were successfully amplified using PCR. The ftfL gene was inserted into the pETduet-1 vector by digestion with BamHI and HindIII, while the mtdA-fchA fragment was inserted by digestion with NdeI and KpnI. The resulting plasmid was transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as indicated by the expected band sizes in gel electrophoresis.

Figure 4: Transformation plate of pETDue-ftfL-mtdA-fchA (A); Enzyme digestion verification for DH5α: pETDue-ftfL F (B), pETDue-ftfL-mtdA-fchA (C); Sequencing results of pETDue-ftfL-mtdA-fchA (D).

The pRSFduet-metF-folA, pETDuet-ftfL-mtdA-fchA plasmid was transformed into E. coli BL21(DE3) to evaluate the co-expression of the ftfL, mtdA, and fchA genes. Protein expression was induced using IPTG and analysed via SDS-PAGE and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the FtfL, MtdA, and FchA proteins, particularly under induction at 37°C. Western Blot analysis confirmed the successful expression of all three proteins, demonstrating effective co-expression.

Figure 5: Expression of metF, folA, mtdA, fchA, and ftfL Proteins in BL21(DE3) Analyzed by SDS-PAGE (up) and Western Blot (down)

A one-step growth curve was generated to compare the growth rates of the different strains. The control strain, BL21, initially entered a rapid growth phase, transitioning into the stationary phase after approximately 10 hours. In contrast, the strains pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A (which contains both the pRSF and pET plasmids) exhibited slower initial growth rates compared to the control strain. Strain A continued growth even at the 12-hour mark, indicating a prolonged exponential growth phase. This suggests that Strain A may have a higher potential for sustained growth, likely due to the combined effects of both plasmids, which enhance L-5-MTHF production and lead to a more robust metabolic capacity under the given conditions.

Figure 6: One-Step Growth Curve for BL21, pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A.