Difference between revisions of "Part:BBa K5301008"

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<p>We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1). </p>
 
<p>We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1). </p>
  
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We used SDS-PAGE to test whether the protein containing mCherry[1-10] had been expressed successfully(Figure 2). The molecular weight of SCSdC-mCh[1-10] (containing mCherry[1-10]) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kDa (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing mCherry[1-10].
 
We used SDS-PAGE to test whether the protein containing mCherry[1-10] had been expressed successfully(Figure 2). The molecular weight of SCSdC-mCh[1-10] (containing mCherry[1-10]) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kDa (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing mCherry[1-10].
  
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Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.
 
Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.
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Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.
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===Sequence and Features===
  
 
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Latest revision as of 11:47, 1 October 2024

MCherry [1-10] is a part of bimolecular fluorescence complementation (BiFC) system.

MCherry [1-10] is a part of bimolecular fluorescence complementation (BiFC) system which could visualize protein interactions. The combination of mCherry [1-10] and mCherry [11] can form the fluorescence of mCherry. With its advantages of a short maturation time and brilliant fluorescence, the mCherry BiFC system could find particular applications for analyzing protein–protein interactions especially in living cells [1].

Usage and Biology

We devised the fusion expression of mCherry[1-10] with spNW15, SpyCatcher and SdyCatcher (namely SCSdC-mCh[1-10]). We used mCherry[1-10] and mCherry[11] parts to emit fluorescence to verify the success of protien connection. If SCSdC-mCh[1-10] can connect to SnTST- mCh[11] successfully, the mCherry[1-10] and mCherry[11] parts can emit fluorescence as a characterization.

We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1).

spc-mche1-10-nw15-mche11-spt.png

Figure 1.The structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag as predicted by AlphaFold

Characterization

We used SDS-PAGE to test whether the protein containing mCherry[1-10] had been expressed successfully(Figure 2). The molecular weight of SCSdC-mCh[1-10] (containing mCherry[1-10]) is 76.3 kDa. And we purified the target protein with a molecular weight of approximately 70-100 kDa (Lane 4 - Lane 5), which demonstrated that we had successfully expressed the protein containing mCherry[1-10].

sds-page-result-of-sdycatcher-for-sdycatcher.png

Figure 2.SDS-PAGE analysis of the extraction results of SCSdC-mCh[1-10] (containing mCherry[1-10]).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.

Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.

mcherry-yingguang.png

Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 31
    Illegal PstI site found at 230
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 31
    Illegal PstI site found at 230
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 31
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 31
    Illegal PstI site found at 230
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 31
    Illegal PstI site found at 230
  • 1000
    COMPATIBLE WITH RFC[1000]


  1. ↑ Fan, J., et al., Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells. Biochemical and Biophysical Research Communications, 2008. 367(1): p. 47-53.