Difference between revisions of "Part:BBa K5301017"
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===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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mSA is used for binding to biotinylated proteins, and sGFP1-10 and sGFP11 self-assemble to form GFP, thereby creating a protein dimer that can report the outcome with fluorescence.
  
 
===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 conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 1, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.
  
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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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https://static.igem.wiki/teams/5301/parts/sgfp1-10-pcr.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.PCR result of the sGFP1-10 tether.The base sequence length of the sGFP1-10 tether is 1071 base pairs (bp)
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K5301017 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5301017 SequenceAndFeatures</partinfo>
  

Revision as of 06:48, 1 October 2024


sGFP1-10 tether is composed of mSA, a 3C linker, a 6His tag, and sGFP 1-10.

sGFP1-10 tether is composed of mSA, a 3C linker, a 6�His tag, and sGFP 1-10.We construct membrane protein dimers through the interaction between biotin and streptavidin, as well as the self-assembly mechanism of sGFP.

Usage and Biology

mSA is used for binding to biotinylated proteins, and sGFP1-10 and sGFP11 self-assemble to form GFP, thereby creating a protein dimer that can report the outcome with fluorescence.

Characterization

We conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 1, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.

sgfp1-10-pcr.png

Figure 1.PCR result of the sGFP1-10 tether.The base sequence length of the sGFP1-10 tether is 1071 base pairs (bp)

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 82
    Illegal AgeI site found at 142
  • 1000
    COMPATIBLE WITH RFC[1000]