Difference between revisions of "Part:BBa K4122017"
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<partinfo>BBa_K4122017 short</partinfo> | <partinfo>BBa_K4122017 short</partinfo> | ||
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SP-GFP-His-Spytag+ SP-RFP-cMyc-Snooptag+ SP-CBM-SC-SC-SNC-SC-V5-7813 | SP-GFP-His-Spytag+ SP-RFP-cMyc-Snooptag+ SP-CBM-SC-SC-SNC-SC-V5-7813 | ||
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<h3>Characterization-Introduction of Tag-Catcher system to co-display PETase and MHETase</h3> | <h3>Characterization-Introduction of Tag-Catcher system to co-display PETase and MHETase</h3> | ||
+ | <p>This year, we replaced the promotor and terminator of GFP on the basis of last year. Promoter-FBA1 and Terminator-ADH2 would achieve better effect and orthogonality. And we used GFP and RFP as markers of successful construction of spycatcher and spytag syetem.</p> | ||
<P>To attain co-display, we combined our display system with two selective protein binding systems, SpyTag-SpyCatcher and SnoopTag-SnoopCatcher. | <P>To attain co-display, we combined our display system with two selective protein binding systems, SpyTag-SpyCatcher and SnoopTag-SnoopCatcher. | ||
In our experiment, GFP and RFP were used to indicate the successful construction of Spycatcher/Spytag and Snoopcatcher/Snooptag systems. We initially tried two catcher types with a ratio of 1:3.</P> | In our experiment, GFP and RFP were used to indicate the successful construction of Spycatcher/Spytag and Snoopcatcher/Snooptag systems. We initially tried two catcher types with a ratio of 1:3.</P> | ||
− | <img src="https://static.igem.wiki/teams/4122/wiki/ | + | <img src="https://static.igem.wiki/teams/4122/wiki/parts/08-the-construction-of-plasmid-ts-pgapdh-teno1a-the-surface-display-system-for-displaying-both-gfp-and-rfp.png" style = "width:55%;"> |
<br/><b>Fig.1</b> The construction of plasmid Ts-PGAPDH--TENO1A, the surface display system for displaying both GFP and RFP. (<a href="https://parts.igem.org/Part:BBa_K4122017">BBa_K4122017</a>) | <br/><b>Fig.1</b> The construction of plasmid Ts-PGAPDH--TENO1A, the surface display system for displaying both GFP and RFP. (<a href="https://parts.igem.org/Part:BBa_K4122017">BBa_K4122017</a>) | ||
<p>SC: Spycatcher BBa_K4122008; SNC: Snoopcatcher BBa_K4122010; V5: V5 tag BBa_K3829004; CBM: carbohydrate binding domain BBa_K4122006</p> | <p>SC: Spycatcher BBa_K4122008; SNC: Snoopcatcher BBa_K4122010; V5: V5 tag BBa_K3829004; CBM: carbohydrate binding domain BBa_K4122006</p> | ||
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+ | <p>GFP+ and RFP+ suggested the successful construction of Spycatcher/Spytag system and Snoopcatcher/Snooptag system.</p> | ||
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+ | <img src="https://static.igem.wiki/teams/4122/wiki/parts/09-the-fluorescence-result-of-the-spy-snoop-tag-and-catcher-system.png | ||
+ | " style = "width:60%;"> | ||
+ | <br/><b>Fig.2</b> The fluorescence result of the spy/snoop tag and catcher system. | ||
+ | <p>A and D, bright field; B and E, Green fluorescence; C and F, Red fluorescence</p> | ||
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+ | <h3>References</h3> | ||
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+ | <p>[1] Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods, 1: 100014 (2021).</p > | ||
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+ | <p>[2] Chengxin Zhang, Peter L. Freddolino, and Yang Zhang. COFACTOR: improved protein function prediction by combining structure, sequence and protein-protein interaction information. Nucleic Acids Research, 45: W291-299 (2017).</p > | ||
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+ | <p>[3] Jianyi Yang, Yang Zhang. I-TASSER server: new development for protein structure and function predictions, Nucleic Acids Research, 43: W174-W181, 2015. </p > | ||
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+ | <p>[4] Lu, Hongyuan, et al. "Machine learning-aided engineering of hydrolases for PET depolymerization." Nature 604.7907 (2022): 662-667.</p > | ||
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Latest revision as of 14:48, 8 October 2022
SP-GFP-His-Spytag+SP-RFP-cMyc-Snooptag+SP-CBM-SC-SC-SNC-SC-V5-7813
SP-GFP-His-Spytag+ SP-RFP-cMyc-Snooptag+ SP-CBM-SC-SC-SNC-SC-V5-7813
This part is composed of three parts:
Part 1:SP-GFP-His-Spytag BBa_K4122014
Part 2:SP-RFP-cMyc-Snooptag BBa_K4122015
Part 3:SP-CBM-SC-SC-SNC-SC-V5-7813 BBa_K4122016
Characterization-Introduction of Tag-Catcher system to co-display PETase and MHETase
This year, we replaced the promotor and terminator of GFP on the basis of last year. Promoter-FBA1 and Terminator-ADH2 would achieve better effect and orthogonality. And we used GFP and RFP as markers of successful construction of spycatcher and spytag syetem.
To attain co-display, we combined our display system with two selective protein binding systems, SpyTag-SpyCatcher and SnoopTag-SnoopCatcher. In our experiment, GFP and RFP were used to indicate the successful construction of Spycatcher/Spytag and Snoopcatcher/Snooptag systems. We initially tried two catcher types with a ratio of 1:3.
Fig.1 The construction of plasmid Ts-PGAPDH--TENO1A, the surface display system for displaying both GFP and RFP. (BBa_K4122017)
SC: Spycatcher BBa_K4122008; SNC: Snoopcatcher BBa_K4122010; V5: V5 tag BBa_K3829004; CBM: carbohydrate binding domain BBa_K4122006
GFP+ and RFP+ suggested the successful construction of Spycatcher/Spytag system and Snoopcatcher/Snooptag system.
Fig.2 The fluorescence result of the spy/snoop tag and catcher system.
A and D, bright field; B and E, Green fluorescence; C and F, Red fluorescence
References
[1] Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods, 1: 100014 (2021).
[2] Chengxin Zhang, Peter L. Freddolino, and Yang Zhang. COFACTOR: improved protein function prediction by combining structure, sequence and protein-protein interaction information. Nucleic Acids Research, 45: W291-299 (2017).
[3] Jianyi Yang, Yang Zhang. I-TASSER server: new development for protein structure and function predictions, Nucleic Acids Research, 43: W174-W181, 2015.
[4] Lu, Hongyuan, et al. "Machine learning-aided engineering of hydrolases for PET depolymerization." Nature 604.7907 (2022): 662-667.
Sequence and Features