Difference between revisions of "Part:BBa K5335029"

Revision as of 16:31, 30 September 2024

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: 无标题文档

Figure 1. Agarose gel electrophoresis image of colony PCR products (target band at 2200 bp)
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Figure 2. SDS-PAGE gel electrophoresis image.
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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: 无标题文档
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, showing.
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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

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 ===Experimental Verification===
 <p style="line-height:1.5rem;">
-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.
+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: <b>BBa_K5335025</b>.
 </p>
 <br>
@@ Line 45: / Line 45: @@
 <br>
 <p style="line-height:1.5rem;">
-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.
+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: <b>BBa_K5335027<b/>.
 </p>
 <br>
@@ Line 69: / Line 69: @@
 <center><img src="https://static.igem.wiki/teams/5335/plant-cpps/15.png" style="width:70%; "></center>
 <br>
-<center><b>Figure 6. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots.
+<center><b>Figure 5. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots.
 <br>
 (A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield. </b> </center>