Difference between revisions of "Part:BBa K5301002"

(→‎Usage and Biology)
 
(7 intermediate revisions by 2 users not shown)
Line 11: Line 11:
 
We devised the fusion expression of SdyTag with spNW15 and SnoopCatcher (namely SnCSdT). We employed SdyTag and SdyCatcher to link two proteins - SCSdC-mCh[1-10] and SnCSdT.
 
We devised the fusion expression of SdyTag with spNW15 and SnoopCatcher (namely SnCSdT). We employed SdyTag and SdyCatcher to link two proteins - SCSdC-mCh[1-10] and SnCSdT.
  
−
===Characterization===
+
<p>We also employed AlphaFold to predict the structure of the protein constituted by SdyTag, spNW15, and SdyCatcher, and discovered that the SdyTag was successfully bound to SdyCatcher(Figure 1). </p>
  
−
We used SDS-PAGE to test whether the protein containing SdyTag had been expressed successfully(Figure 1). The molecular weight of SnCSdT (containing SdyTag) is 42.1 kDa. And we purified the target protein with a molecular weight of approximately 43 kD (Lane 5 - Lane 8), which demonstrated that we had successfully expressed the protein containing SdyTag.
 
 
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
 
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
 +
https://static.igem.wiki/teams/5301/parts/sdt-sdc-nw15.png
 +
</div><div class="thumbcaption">
 +
Figure 1.The structure of the protein constituted by SdyTag, spNW15 and SdyCatcher as predicted by AlphaFold
 +
</div></div></div></div>
 +
 +
===Characterization===
 +
 +
We used SDS-PAGE to test whether the protein containing SdyTag had been expressed successfully(Figure 2). The molecular weight of SnCSdT (containing SdyTag) is 42.1 kDa. And we purified the target protein with a molecular weight of approximately 43 kD (Lane 5 - Lane 8), which demonstrated that we had successfully expressed the protein containing SdyTag.
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.7;overflow:hidden;">
 
https://static.igem.wiki/teams/5301/parts/sds-page-result-of-sncsdt-for-sdytag.png
 
https://static.igem.wiki/teams/5301/parts/sds-page-result-of-sncsdt-for-sdytag.png
 
</div><div class="thumbcaption">
 
</div><div class="thumbcaption">
−
Figure 1.SDS-PAGE analysis of the extraction results of SnCSdT (containing SdyTag).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 SnCSdT-M15-GS is 42.1 kDa.
+
Figure 2.SDS-PAGE analysis of the extraction results of SnCSdT (containing SdyTag).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 SnCSdT-M15-GS is 42.1 kDa.
 
</div></div></div></div>
 
</div></div></div></div>
 +
 +
===Sequence and Features===
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
<span class='h3bb'></span>
 
<partinfo>BBa_K5301002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5301002 SequenceAndFeatures</partinfo>
  

Latest revision as of 09:42, 1 October 2024


SdyTag can achieve covalent binding of proteins through Tag-Catcher interaction.

SdyTag is a fibronectin-binding protein. It can cooperate with SdyCatcher to achieve covalent binding of proteins through Tag-Catcher interaction. SdyTag and SdyCatcher can also be used alongside SpyTag-SpyCatcher partners, to demonstrate kinetically controlled directed protein ligation, domain specific protein circularization and macromolecular assembly [1].

Usage and Biology

We devised the fusion expression of SdyTag with spNW15 and SnoopCatcher (namely SnCSdT). We employed SdyTag and SdyCatcher to link two proteins - SCSdC-mCh[1-10] and SnCSdT.

We also employed AlphaFold to predict the structure of the protein constituted by SdyTag, spNW15, and SdyCatcher, and discovered that the SdyTag was successfully bound to SdyCatcher(Figure 1).

sdt-sdc-nw15.png

Figure 1.The structure of the protein constituted by SdyTag, spNW15 and SdyCatcher as predicted by AlphaFold

Characterization

We used SDS-PAGE to test whether the protein containing SdyTag had been expressed successfully(Figure 2). The molecular weight of SnCSdT (containing SdyTag) is 42.1 kDa. And we purified the target protein with a molecular weight of approximately 43 kD (Lane 5 - Lane 8), which demonstrated that we had successfully expressed the protein containing SdyTag.

sds-page-result-of-sncsdt-for-sdytag.png

Figure 2.SDS-PAGE analysis of the extraction results of SnCSdT (containing SdyTag).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 SnCSdT-M15-GS is 42.1 kDa.

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]


  1. ↑ Tan, L.L., S.S. Hoon and F.T. Wong, Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLOS ONE, 2016. 11(10): p. e0165074.