Part:BBa_K5335029

Spycatcher-CPPs(R9-Tag)

The SpyCatcher-CPPs system, designed and employed in our team project, serves as a versatile tool for validating project feasibility and aiding in the characterization of other components. Originating from the CnaB2 domain of Streptococcus pyogenes fibronectin-binding protein, this system comprises a 12 kDa SpyCatcher protein and a 13-residue SpyTag peptide. The spontaneous formation of an isopeptide bond between SpyC and SpyT enables the efficient delivery of target proteins, fused with SpyTag and the cell-penetrating peptide R9-Tag, into plant cells.

Experimental Verification

The construction and validation of the SpyCatcher component in this part involved experiments such as colony PCR, SDS-PAGE, and Western blotting. Detailed procedures for the validation can be found in the part: BBa_K5335025.

Here are some of the results:

Figure 1. Agarose gel electrophoresis image of colony PCR products (target band at 2200 bp)

Figure 2. SDS-PAGE gel electrophoresis image.

Figure 3. Western Blot Image.

The construction and validation of the CPPs(R9-Tag) component in this part involved the experiment,detection of plant cell penetration by AmCyan-CPPs fusion protein using laser scanning confocal microscopy. Detailed procedures for the validation can be found in the part: BBa_K5335027.

Here are some of the results:
Figure 4. Confocal microscopy images of root tissues.
(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield. (D) High-magnification view of root hair cells in the merged image.

Figure 5. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots.
(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield.

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]

Functional Parameters