Difference between revisions of "Part:BBa K2132003"
 
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<partinfo>BBa_K2132003 short</partinfo>
 
<partinfo>BBa_K2132003 short</partinfo>
  
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This is a reporter protein for any protein with SpyCatcher tagged. mCherry-SpyTag is stable under room temperature. It can bind to SpyCatcher within 20 min.
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This is a reporter protein for any protein containing SpyCatcher domain. mCherry-SpyTag is stable under room temperature. It can bind to SpyCatcher within 20 mins at room temperature. In our project, we used it to check if expression of CsgA-SpyCatcher in biofilm form is successful. Regarding the principle of Spytag and Spycatcher, please refer to the two references at the bottom.
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In our project, we used it to see if the expression of CsgA-SpyCatcher is successful.
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<img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" width="100%" height="100%">
 
<img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" width="100%" height="100%">
 
<figcaption>
 
<figcaption>
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<b>Fig. 1</b>:The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria.. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.
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<b>Fig. 1</b>:The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.
 
</figcaption>
 
</figcaption>
 
</figure>
 
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</p>
 
</p>
 
<h3>Reference</h3>
 
<h3>Reference</h3>
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<p>Bijan Zakeria, J. O.-L. (2012, February 24). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. PNAS. </p>
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<p>B. Zakeri et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012). </p>
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<p>Zsofia Botyanszki, 1. P. (2015, May 20). Engineered Catalytic Biofilms: Site-Specific Enzyme Immobilization onto E. coli Curli Nanofibers. Biotechnology and Bioengineering. </p>
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<p>Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, N. S. Joshi, Engineered catalytic biofilms: Site‐specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology and bioengineering 112, 2016-2024 (2015).
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</p>

Latest revision as of 12:33, 19 October 2016


mCherry-SpyTag

This is a reporter protein for any protein containing SpyCatcher domain. mCherry-SpyTag is stable under room temperature. It can bind to SpyCatcher within 20 mins at room temperature. In our project, we used it to check if expression of CsgA-SpyCatcher in biofilm form is successful. Regarding the principle of Spytag and Spycatcher, please refer to the two references at the bottom.


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]

Characterization

Experiments were conducted to characterize this biobrick:

  • Fluoresence Binding Test

Fluoresence Binding Test

Fig. 1:The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.

As figure illustrated, his-CsgA-SpyCatcher-his mutant incubated with mcherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicating the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on E.coli. That to a large extent prove the specificity of our desired linkage between SpyTag and SpyCatcher system.

Reference

B. Zakeri et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012).

Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, N. S. Joshi, Engineered catalytic biofilms: Site‐specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology and bioengineering 112, 2016-2024 (2015).