Part:BBa_K4122015

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.

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 BBa_K4122015 SequenceAndFeatures