Difference between revisions of "Part:BBa K5301008"
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We devised the fusion expression of mCherry[1-10] with spNW15, SpyCatcher and SdyCatcher (namely SCSdC-mCh[1-10]). | 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 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. | ||
| + | |||
| + | <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> | ||
| + | |||
| + | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.25;overflow:hidden;"> | ||
| + | https://static.igem.wiki/teams/5301/parts/spc-mche1-10-nw15-mche11-spt.png | ||
| + | </div><div class="thumbcaption"> | ||
| + | Figure 1.The structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag as predicted by AlphaFold | ||
| + | </div></div></div></div> | ||
===Characterization=== | ===Characterization=== | ||
| − | We used SDS-PAGE to test whether | + | 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]. |
| − | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0. | + | |
| − | https://static.igem.wiki/teams/5301/parts/sds-page-of- | + | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.6;overflow:hidden;"> |
| + | https://static.igem.wiki/teams/5301/parts/sds-page-result-of-sdycatcher-for-sdycatcher.png | ||
</div><div class="thumbcaption"> | </div><div class="thumbcaption"> | ||
| − | + | 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. | |
</div></div></div></div> | </div></div></div></div> | ||
| + | |||
| + | 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. | ||
| + | |||
| + | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.9;overflow:hidden;"> | ||
| + | https://static.igem.wiki/teams/5301/parts/mcherry-yingguang.png | ||
| + | </div><div class="thumbcaption"> | ||
| + | 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. | ||
| + | </div></div></div></div> | ||
| + | |||
| + | ===Sequence and Features=== | ||
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<partinfo>BBa_K5301008 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5301008 SequenceAndFeatures</partinfo> | ||
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).
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].
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.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 31
Illegal PstI site found at 230 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 31
Illegal PstI site found at 230 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 31
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 31
Illegal PstI site found at 230 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 31
Illegal PstI site found at 230 - 1000COMPATIBLE WITH RFC[1000]
- ↑ 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.