Part:BBa_K5335025

SpyCatcher-VDAL-CPPs (R9-Tag)

To enhance plant immunity, a novel fusion protein, SpyCatcher-VDAL-CPPs, was designed. This construct incorporates a SpyCatcher tag_[1] for conjugation, VDAL for immune activation_[2], and the R9 cell-penetrating peptide for intracellular delivery_[3]. Functional studies demonstrated the capacity of this fusion protein to penetrate plant cell membranes and induce an ETI response. Molecular characterization and DAB staining confirmed the successful construction and immunogenic activity of the fusion protein.
The design was verified as shown in Figure 1.

Figure 1. Experimental circuit design diagram

Usage and Biology

VDAL, an Aspf2-like protein derived from Verticillium dahliae, has been shown to activate PTI responses when expressed extracellularly _[4] and can also induce endogenous ETI immune responses when expressed intracellularly. To verify the function of the constructed circuit, a pET28a-based expression system was employed, with the T₇ promoter under the control of the lac operon. The circuit includes the engineered SpyCatcher-VDAL-CPPs protein, SpyTag-6*His protein, and SpyCatcher-CPPs protein. The SpyCatcher-SpyTag (SpyC/SpyT) system is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein. SpyC is an immunoglobulin-like protein with a molecular weight of approximately 12 kDa, while SpyT is a short peptide consisting of 13 residues. These two components can specifically recognize each other and spontaneously form an isopeptide bond, enabling high-affinity binding. The designers aimed to utilize this covalent linkage during co-expression to verify the functionality of the SpyCatcher-SpyTag system and facilitate the purification of the target protein.
The constructed plasmid vector is illustrated in Figure 2.

Figure 2. Plasmid Vector

Experimental Verification

Transformation

After chemical transformation using calcium chloride, the constructed plasmid was introduced into E. coli BL21 (DE3). The transformed cells were plated on LB agar plates supplemented with kanamycin and incubated at 37℃ for 16 hours. Colony PCR was performed on individual colonies to verify the presence of the plasmid.
The results of the colony PCR are presented in Figure 3.

Figure 3. Agarose gel electrophoresis image of colony PCR products (target band at 2200 bp) After sequencing the selected single colonies and confirming the results, the researchers proceeded with subsequent experiments.

SDS-PAGE

Figure 2. Plasmid Vector

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]