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: <b>BBa_K5335025</b>.
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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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<partinfo>BBa_K5335029 SequenceAndFeatures</partinfo>
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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: <b>BBa_K5335027</b>.
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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.</b>
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(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield.
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(D) High-magnification view of root hair cells in the merged image. </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 5. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots.</b>
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(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield.</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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Latest revision as of 21:42, 1 October 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.

无标题文档

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