Conjugation

Part:BBa_K2976010

Designed by: Jiatong Chen   Group: iGEM19_CPU_CHINA   (2019-08-04)
Revision as of 00:26, 21 October 2019 by NF-kB pathway (Talk | contribs) (→‎Usage and Biology)

Improved T2A

Oligopeptide “2A‐like” sequences are found in a range of RNA virus genomes controlling protein biogenesis and T2A derives from Thosea asigna virus. The 2A peptide consensus motif is extremely rare and is always associated with cleavage activity between the glycine and the proline. The insertion of T2A within a single open reading frame (ORF) produces multiple proteins [1]. Besides, inserting a GSG linker before the T2A sequence significantly improves its self-cleaving efficiency and ensures complete cleavage [2].

Usage and Biology

Some viruses use 2A peptides, or 2A–like sequences, to mediate protein cleavage. Through a ribosomal ‘skip’ mechanism, the 2A consensus motif appears to impair normal peptide bond formation between the glycine and the proline [3]. In addition, inclusion of a Gly-Ser-Gly (GSG) spacer in front of 2A peptide ensures complete ‘cleavage’, so we take this approach to optimize cleavage effectiveness of T2A. In 2019 CPU_CHINA project, we utilize P2A and improved T2A to co-express TLR1:TLR2:CD14 cluster on our designed cell membrane.

In order to confirm the cleavage efficiency of the GSG-added T2A was improved, T2A (Part:BBa_K1993019) and improved T2A (BBa_K2976010) were separately constructed into expression vector for co-expression of TLR1 and CD14. And we performed flow cytometry on cells transfected with plasmid (TLR1-T2A-CD14) and plasmid (TLR1-improved T2A-CD14).


Figure 1. showed that the amount of CD14 in the cells that were transfected with plasmid (TLR1-improved T2A-CD14) was higher than that of the plasmid (TLR1-T2A-CD14), which proved that the cleavage efficiency of the part is enhanced, and verified our improved part achieved the expected functionality.

Reference

[1] Luke, G. A. , Ryan, M. D. (2018). Therapeutic applications of the 'NPGP' family of viral 2As. Rev Med Virol, 28(6):e2001.

[2] Yang, X. , Zeng, Q. , Wang, M. , Cheng, A. , Pan, K. , & Zhu, D. , et al. (2018). Dhav-1 2a1 peptide – a newly discovered co-expression tool that mediates the ribosomal “skipping” function. Frontiers in Microbiology, 9.

[3] Ikegami, T. , Lee, B. , Smith, J. K. , Hill, T. E. , & Park, A. . (2016). Optimized P2A for reporter gene insertion into nipah virus results in efficient ribosomal skipping and wild-type lethality. Journal of General Virology, 97(4), 839-843.


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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Parameters
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