Difference between revisions of "Part:BBa K2132003"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2132003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2132003 SequenceAndFeatures</partinfo>
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<html>
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<h3>Characterization</h3>
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<p>
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Experiments were conducted to characterize this biobrick:
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<ul>
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<li> Fluoresence Binding Test </li>
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</ul>
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</p>
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<h4> Fluoresence Binding Test </h4>
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<figure>
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<img src="https://parts.igem.org/File:Shanghaitechchina_SCH%2BQD.tiff" width="100%" height="100%">
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<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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</figcaption>
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</figure>
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<p>
 
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.  
 
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.  
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</p>
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2132003 parameters</partinfo>
 
<partinfo>BBa_K2132003 parameters</partinfo>
 
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Revision as of 12:26, 13 October 2016


mCherry-SpyTag

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. In our project, we used it to see if the expression of CsgA-SpyCatcher is successful.


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.

===Functional Parameters=== BBa_K2132003 parameters