Difference between revisions of "Part:BBa K1993019"

Revision as of 08:03, 23 July 2021

T2A

T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. It is a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. [1]

In our project, we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Taking advantage of function of T2A, we constructed a plasmid that T2A linked between two protein coding genes. For example, we constructed BBa_K1993006(Luciferase-T2A-dTomato-T2A-hFTH) to ensure luciferase, dTomato and hFTH could be expressed.

References

[1] Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. [J]. Plos One, 2011, 6(4): e18556..

improvement by CPU_CHINA 2019

Figure 1. Flow cytometry analysis of CD14 expression in cells without transfection, transfected with plasmid (TLR1-T2A-CD14) and transfected with plasmid (TLR1-improved T2A-CD14).

In order to confirm the cleavage efficiency of the GSG-added T2A was improved, T2A (BBa_K1993019) and improved T2A (Part: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). According to Figure 1, 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.

Sequence and Features