Engineering

To evaluate the expression efficiency of the FNR and T7 promoters for the genes NSP4-K-Hecate and NSP4-K-Melittin, novel recombinant plasmids demonstrated pET-T7-NSP4-K-Hecate (Fig. 1a) and pET-T7-NSP4-K-Melittin (Fig. 1b), were designed. The pET-T7-CYP450-Flag plasmid was selected as the backbone. The entire plasmid, with the exception of the CYP450-Flag gene, was amplified so as to generate a linearized vector possessing homologous ends designed for seamless ligation with the inserts.

The coding sequences for NSP4-K-Hecate and NSP4-K-Melittin were amplified from pET28a-FNR-NSP4-K-Hecate and pET28a-FNR-NSP4-K-Melittin, respectively.

Figure 1: The illustration of our desired recombinant plasmid. a) Hecate gene in the pET-T7 backbone. b) Melittin gene in the pET-T7 backbone

The initial stage started with PCR amplification to generate the Hecate and Melittin inserts, as well as to linearize the pET-T7 vector backbone for subsequent Gibson Assembly, where the inserts will replace the original CYP450-Flag coding sequence in the plasmid pET-T7-CYP450-Flag.

Analysis of these PCR products by gel electrophoresis demonstrated that amplification for all three fragments—the Hecate insert, the Melittin insert, and the vector backbone—was unsuccessful, as no bands of the expected sizes were observed. Specifically, neither the Melittin nor Hecate inserts produced detectable bands, indicating complete amplification failure. For the pET-T7 backbone, several non-specific bands appeared at approximately 8 kb, 3 kb, and 2 kb, but there was no band at the expected ~5.6 kb size corresponding to the linearized vector.

Figure 2: The DNA gel electrophoresis for the Cloning PCR result of Hecate insert (2), Melittin insert (3) and Vector (5)

The PCR failure may be attributed to suboptimal primer design, as there is a significant difference in melting temperatures (Tm) between the forward and reverse primers. This discrepancy complicates the selection of an appropriate annealing temperature; therefore, we have decided to redesign the primers in the next cycle.

Based on the lessons from Cycle 1, we initiated a comprehensive redesign. New primers are designed for the Hecate insert, Melittin insert, and pET-T7 backbone from scratch using Snapgene software to optimize binding characteristics, hope to obtain successful PCR amplification of all fragments.

The newly designed primers:

Note: The highlighted in red are the overhangs.

Subsequent to the redesign of the primers, we undertook several iterative rounds of PCR optimization in an effort to amplify the three target fragments. Despite the implementation of newly designed primers and verified templates, standard PCR protocols remained unsuccessful. Consequently, we systematically modified key PCR parameters. Annealing temperature was optimized by performing temperature gradient PCR, testing a range of temperatures surrounding the calculated Tm of the new primers to identify the optimal condition. Additionally, the annealing time was significantly increased from the conventional 30 seconds to 1 minute to promote more complete and specific primer-template hybridization.

After adjusting the PCR protocol and the primer, we finally got clear bands for the Hecate insert and Vector, which was confirmed using gel electrophoresis. In the Fig. 3a, we can see bands at the correct position for the Hecate insert fragment (189bp), and in the Fig. 3b, the Vector fragment (5614bp) compared to the original plasmid. We cut these bands and performed gel purification. In an effort to resolve the persistent PCR failure for the Melittin insert, the original pET28a-FNR-NSP4-K-Melittin template plasmid was sequenced; sequencing revealed that the clone obtained from the company was incorrect and did not contain the intended insert.

Figure 3: The DNA gel electrophoresis of the Hecate and Melittin inserts and the vector.

Agarose gel electrophoresis confirmed successful PCR amplification of both the Hecate insert and the pET-T7 backbone; however, all attempts to amplify the Melittin insert consistently failed. Consequently, Gibson Assembly was employed to synthesize the Hecate construct (Fig. 1a). As repeated failures were observed for the Melittin insert, we hypothesized that the issue may reside with the original plasmid template. Therefore, we proceeded to re-extract the plasmid and have submitted it for sequencing analysis.

Sequencing analysis from the previous cycle confirmed a problem with the original clone obtained from the commercial supplier. As a result, we reordered the Melittin plasmid (pET28a-FNR-NSP4-K-Melittin). Consequently, progress on the Melittin recombinant constructs was temporarily halted, while synthesis of the Hecate construct continued without interruption.

Following gel purification, we successfully obtained the DNA fragments required for subsequent Gibson assembly. The assembled products will be verified by agarose gel electrophoresis, after which they will be transformed into BL21 competent cells for plasmid replication. The extracted plasmids will then be submitted for sequencing to confirm the assembly accuracy.

In Fig. 4, DNA gel electrophoresis analysis of the assembled product revealed a faint band at an unexpected position. Despite the suboptimal gel result, the band was gel-purified, transformed into BL21 cells, subjected to plasmid miniprep, and subsequently sequenced. Sequencing results indicated that the assembly had not succeeded, as the recombinant plasmid was not correctly formed.

Figure 4: The DNA gel electrophoresis of the Gibson assembly of the Hecate construct. No.2 stands for the Hecate insert, and No.5 stands for the pET-T7 Vector.