Composite

Part:BBa_K4122015

Designed by: JIAHUA FANG   Group: iGEM22_IvyMaker-China   (2022-09-15)
Revision as of 08:13, 6 October 2022 by Lunazhou (Talk | contribs)


PFBA1-SP-RFP-cMyc-Snooptag-TPGK1

Characterization-Introduction of Tag-Catcher system to co-display PETase and MHETase

In this assembly, RFP is a RED fluorescent protein, showing red fluorescence, cMyc tag is used for protein purification, and Snooptag will form a tight connection with snoopcatcher, which in turn anchors the attached RFP to the cell surface. Promoter-PFBA1,Terminator-TPGK1.

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.

<img src="08-the-construction-of-plasmid-ts-pgapdh-teno1a-the-surface-display-system-for-displaying-both-gfp-and-rfp.png" style = "width:55%;">
Fig.1 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>)

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.

<img src="09-the-fluorescence-result-of-the-spy-snoop-tag-and-catcher-system.png " style = "width:60%;">
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


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]


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