Difference between revisions of "Part:BBa K1993019"

Revision as of 00:34, 21 October 2019

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

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.

For more details, please check out our part improvement page.

Sequence and Features