Difference between revisions of "Part:BBa K5301012"
| (7 intermediate revisions by the same user not shown) | |||
| Line 3: | Line 3: | ||
<partinfo>BBa_K5301012 short</partinfo> | <partinfo>BBa_K5301012 short</partinfo> | ||
| − | + | 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<partinfo>BBa_K5301014</partinfo>)<sup>[1]</sup>. | |
| − | + | 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. | |
| − | |||
| − | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0. | + | ==Characterization== |
| − | https://static.igem.wiki/teams/5301/parts/ | + | |
| + | We performed an in vitro mixing and incubation of sGFP1-10 with sGFP11 to achieve their binding(in the form of sGFP1-10/11 tether<partinfo>BBa_K5301017</partinfo><partinfo>BBa_K5301018</partinfo>). The images captured under a fluorescence microscope are shown in the figure below. | ||
| + | |||
| + | |||
| + | <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/sgfp-fluorescent.png | ||
</div><div class="thumbcaption"> | </div><div class="thumbcaption"> | ||
| − | Figure 1. | + | Figure 1.Fluorescence images following the reassembly of sGFP(4×10).Figure A and Figure C represent the background fluorescence, while Figures B and D show the observations after the addition of the samples at corresponding positions. By comparing these results, the corresponding fluorescence signals are obtained to eliminate false positives. |
</div></div></div></div> | </div></div></div></div> | ||
| Line 23: | Line 27: | ||
<!-- --> | <!-- --> | ||
| − | + | ||
| + | ==Sequence and Features== | ||
| + | |||
<partinfo>BBa_K5301012 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5301012 SequenceAndFeatures</partinfo> | ||
| Line 30: | Line 36: | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K5301012 parameters</partinfo> | <partinfo>BBa_K5301012 parameters</partinfo> | ||
| − | <!-- --> | + | <!-- --> |
| + | |||
| + | References: | ||
| + | [1]Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol. 2005 Jan;23(1):102-7. doi: 10.1038/nbt1044. Epub 2004 Dec 5. PMID: 15580262. | ||
Latest revision as of 06:32, 2 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 (sGFP11BBa_K5301014)[1].
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(in the form of sGFP1-10/11 tetherBBa_K5301017BBa_K5301018). The images captured under a fluorescence microscope are shown in the figure below.
Figure 1.Fluorescence images following the reassembly of sGFP(4×10).Figure A and Figure C represent the background fluorescence, while Figures B and D show the observations after the addition of the samples at corresponding positions. By comparing these results, the corresponding fluorescence signals are obtained to eliminate false positives.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References: [1]Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol. 2005 Jan;23(1):102-7. doi: 10.1038/nbt1044. Epub 2004 Dec 5. PMID: 15580262.