Difference between revisions of "Part:BBa K5335029"

 
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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.
 
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.
  
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===Experimental Verification===
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===Usage and Biology===
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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.
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<b>Here are some of the results:</b>
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<center><img src="https://static.igem.wiki/teams/5335/plant-cpps/3.png" style="width:38%; "></center>
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<center><b>Figure 1. Agarose gel electrophoresis image of colony PCR products (target band at 2200 bp) </b> </center>
  
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<span class='h3bb'>Sequence and Features</span>
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<center><img src="https://static.igem.wiki/teams/5335/plant-cpps/5.png" style="width:70%; "></center>
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<center><b>Figure 2. SDS-PAGE gel electrophoresis image.</b> </center>
  
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<center><img src="https://static.igem.wiki/teams/5335/plant-cpps/6.png" style="width:50%; "></center>
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<center><b>Figure 3. Western Blot Image.</b> </center>
  
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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.
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</p>
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<b>Here are some of the results:</b>
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<center><img src="https://static.igem.wiki/teams/5335/plant-cpps/14.png" style="width:40%; "></center>
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<center><b>Figure 4. Confocal microscopy images of root tissues.
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(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.</b> </center>
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<center><img src="https://static.igem.wiki/teams/5335/plant-cpps/15.png" style="width:70%; "></center>
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<center><b>Figure 6. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots.
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(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield. </b> </center>
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===Sequence and Features===
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<partinfo>BBa_K5335029 SequenceAndFeatures</partinfo>
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K5335029 parameters</partinfo>
 
<partinfo>BBa_K5335029 parameters</partinfo>
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Revision as of 16:29, 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)

无标题文档

Figure 2. SDS-PAGE gel electrophoresis image.

无标题文档

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.

无标题文档

Figure 6. 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