Difference between revisions of "Part:BBa K5301012"
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<partinfo>BBa_K5301012 short</partinfo>
 
<partinfo>BBa_K5301012 short</partinfo>
  
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Constructing membrane protein dimers using the self-assembly of GFP1-10 and GFP11.
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The Split-GFP system is a biomolecular tool based on green fluorescent protein (GFP) for studying protein-protein interactions, protein localization, formation of protein complexes, and a variety of other biological processes. It divides GFP into two non-fluorescent fragments, typically including a large fragment (sGFP1-10) and a small fragment (sGFP11).
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sGFP1-10 represents the first 10 beta-strands of GFP. This fragment is non-fluorescent on its own, but when it interacts with the GFP11 fragment, it can reassemble into a complete GFP structure with fluorescent activity. sGFP1-10 is usually overexpressed in cells to interact with the GFP11 fragment fused to the target protein.When these two fragments come close to or interact with each other, they can reassemble into the complete GFP structure, thereby restoring fluorescent activity
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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We devised the fusion expression of SdyCatcher with spNW15, SpyCatcher and mCherry[1-10] (namely SCSdC-mCh[1-10]). We employed SdyTag and SdyCatcher to link two proteins - SCSdC-mCh[1-10] and SnCSdT.
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The Split-GFP system has a variety of applications, including real-time monitoring of protein-protein interactions, visualization of protein localization, and the study of protein complex formation. In our team's experiments, sGFP1-10 and sGFP11 are individually fused and expressed with the target proteins. The self-assembly of sGFP1-10 and sGFP11 into a complete GFP with fluorescent activity results in the formation of a dimer.
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===Characterization===  
 
===Characterization===  
  
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We used SDS-PAGE to test whether the protein containing SdyCatcher had been expressed successfully(Figure 1). The molecular weight of SCSdC-mCh[1-10] (containing SdyCatcher) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kD (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing SdyCatcher.
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We performed an in vitro mixing and incubation of sGFP1-10 with sGFP11 to achieve their binding. The images captured under a fluorescence microscope are shown in the figure below.
  
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
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https://static.igem.wiki/teams/5301/parts/sds-page-result-of-sdycatcher-for-sdycatcher.png
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.25;overflow:hidden;">
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https://static.igem.wiki/teams/5301/parts/sgfp-fluorescent.png
 
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Figure 1.SDS-PAGE analysis of the extraction results of SCSdC-mCh[1-10] (containing SdyCatcher).The gel was run at 80 V for 10 minutes and then at 150 V for 20 minutes, followed by staining with Coomassie Brilliant Blue. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa.
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Figure 1.Fluorescence images following the reassembly of sGFP.
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
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<partinfo>BBa_K5301012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5301012 SequenceAndFeatures</partinfo>
  

Revision as of 06:09, 1 October 2024


sGFP1-10 is an engineered version of the first 10 strands of the GFP beta-barrel.

The Split-GFP system is a biomolecular tool based on green fluorescent protein (GFP) for studying protein-protein interactions, protein localization, formation of protein complexes, and a variety of other biological processes. It divides GFP into two non-fluorescent fragments, typically including a large fragment (sGFP1-10) and a small fragment (sGFP11).

sGFP1-10 represents the first 10 beta-strands of GFP. This fragment is non-fluorescent on its own, but when it interacts with the GFP11 fragment, it can reassemble into a complete GFP structure with fluorescent activity. sGFP1-10 is usually overexpressed in cells to interact with the GFP11 fragment fused to the target protein.When these two fragments come close to or interact with each other, they can reassemble into the complete GFP structure, thereby restoring fluorescent activity

Usage and Biology

The Split-GFP system has a variety of applications, including real-time monitoring of protein-protein interactions, visualization of protein localization, and the study of protein complex formation. In our team's experiments, sGFP1-10 and sGFP11 are individually fused and expressed with the target proteins. The self-assembly of sGFP1-10 and sGFP11 into a complete GFP with fluorescent activity results in the formation of a dimer.


Characterization

We performed an in vitro mixing and incubation of sGFP1-10 with sGFP11 to achieve their binding. The images captured under a fluorescence microscope are shown in the figure below.


sgfp-fluorescent.png

Figure 1.Fluorescence images following the reassembly of sGFP.


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]