Goal: To express and purify three distinct proteins: T3 phage tail fiber protein (TFP), the tailspike protein (TSP), and the cell wall binding domain (CBD) of the endolysin plyV12, we aim to construct three expression plasmids: pET28a-gp17, pET28a-Tailspike and pET28a-plyV12. These plasmids will be designed to facilitate the efficient expression of the respective proteins in BL21(DE3) bacterial, thereby enabling subsequent purification and functional studies.
1) Gene and Linearized Vector Amplification
To construct the pET28a-Tailspike, pET28a-gp17, and pET28a-plyV12 plasmids, we first amplified the target genes and linearized pET28a vector fragments using Polymerase Chain Reaction (PCR). The PCR products were then analyzed by agarose gel electrophoresis to verify the amplification and size of the DNA fragments.
The agarose gel electrophoresis results demonstrated successful amplification of the target genes and the linearized vector fragments. Distinct bands corresponding to the expected sizes of the tailspike (2067 bp), gp17(1689 bp), and plyV12(525 bp) genes were observed, confirming the presence of the desired PCR products (Figure 1). Additionally, the linearized pET28a vector fragment (5854 bp) was also clearly visible, indicating successful linearization.
These results indicate that the PCR amplification of the target genes and the linearized pET28a vector fragments were successful, providing the necessary components for subsequent cloning steps.
Figure 1. Agarose Gel Electrophoresis of PCR Amplified Products for Plasmid Construction
Agarose gel electrophoresis (1% agarose) of PCR amplified products used for the construction of plasmids. PCR product of the (A) gp17 gene and corresponding linearized pET28a vector. (B) gp17 gene and corresponding linearized pET28a vector. (C) Tailspike gene and corresponding linearized pET28a vector. Note: M indicated DNA ladder.
2) Gibson Assembly and Bacterial Transformation
Following the successful amplification of the target genes and linearized vector fragments by PCR, we proceeded with the Gibson Assembly to recombine the DNA fragments into the desired plasmids: pET28a-Tailspike, pET28a-gp17, and pET28a-plyV12. The Gibson Assembly reaction mixtures were then used to transform DH5α competent cells.
After transformation, the DH5α cells were plated on LB agar plates containing kanamycin, for selection. Numerous single colonies were observed after the plates were incubated overnight at 37°C (Figure 2), indicating successful transformation.
Figure 2: Bacterial Colony Formation on LB Agar Plates Post-Transformation
LB agar plates containing kanamycin selection were used to assess the success of the Gibson Assembly and transformation into DH5α cells. The plates were incubated overnight at 37°C. Plate showing colonies resulting from the transformation with (A) the pET28a-gp17 plasmid. (B) the pET28a-plyV12 plasmid. (C) the pET28a-Tailspike plasmid.
3)Colony PCR Screening
To identify potential positive clones, colony PCR was performed on several bacterial colonies from each transformation plate. The colony PCR products were analyzed via agarose gel electrophoresis. The gel images revealed distinct bands corresponding to the expected sizes of the Tailspike, gp17, and plyV12 gene inserts (Figure 3A), indicating the presence of the desired recombinant plasmids in several colonies.
These colonies will be further verified by sequencing. The sequencing results confirmed that the gene sequences in the plasmids were correct and matched the expected sequences for gp17, plyV12, and Tailspike (Figure 3B). This confirmed that the plasmid constructs pET28a-gp17, pET28a-plyV12, and pET28a-tailspike were successfully constructed without any mutations.
Figure 3: Colony PCR and Sequencing Results for the Identification of Positive Clones
(A) Agarose gel electrophoresis showing colony PCR results for the identification of positive clones containing the recombinant plasmids. Distinct bands at the expected sizes for the gp17, Tailspike and plyV12 inserts confirm the presence of the desired recombinant plasmids.
(B) Sequencing chromatograms of representative positive clones. The chromatograms show high-quality sequences with no mutations or insertions.