Difference between revisions of "Part:BBa K2976009"

Revision as of 15:51, 21 October 2019

NF-κB induced promoter

Usage

After the activation of the NF-κB transcriptional factor, and with an NF-κB inducible promoter to responsively promote downstream pathway designed, the normol cells can release granulysin to destroy Mtb in the blood circulation; in the meantime, they can secret targeting exosomes containing miRNA to rescue the infected macrophages.

Biology

Granulysin is a member of the saposin-like protein (SAPLIP) 3 family, which has a broad spectrum of antimicrobial activity and can kill the intracellular pathogen M.tuberculosis in macrophages with the assistance of perforin. The targeting exosomes containing miRNA let-7f to kill intracellular bacteria can conquer the drawback of granulysin. Exogenous supplementation of let-7f increases the level of endogenous let-7f and further up regulates NF-κB, then the secretion of cytokines, chemokines and NO which can kill intracellular Mtb are boosted.

Characterization

2019 CPU_CHINA attempted to develop a novel strategy to treat tuberculosis based on immune-like cells. Since granulysin is a major part of our project, we conducted research on it and obtained valuable results.

Figure 1.Induced secretion of granulysin.

Plasmid (TLR2-P2A-TLR1-T2A-CD14) and plasmid (NF-κB induced promoter-granulysin) were co-transfected into HEK293T cells. Then, we utilized Pam3Cys-Ser-(Lys)4, TLR1/TLR2 agonist to stimulate Toll like receptor (TLR) and its downstream signaling pathway. In our project, the activation of NF-κB can eventually leads to the expression of granulysin. Since the granulysin contains signal peptides, it is eventually destined to be secreted out of cells. Therefore, we determine the concentration of granulysin by ELISA assay. As shown in Figure 1, the concentration of granulysin in the cell culture medium is significantly higher than other groups at 24h (A), 36h (B) and 48h (C).

Figure 2.Time-Killing Curve of granulysin against M. smegmatis.

Since M.tuberculosis is quite a dangerous species, we utilize a safer strain, M.smegmatis as a substitute to test in vitro antimicrobial activity of granulysin. The same amount of M.smegmatis was seeded into the cell culture medium with or without granlysin. Then, bacteria and cell medium were incubated at 37℃ with 200 rpm and OD600 of the medium was determined every 10 hours. The results demonstrate that granulysin exhibits good antimicrobial activity against M.smegmatis.

For more details, please check out our demonstrate page.

References

[1] Stenger, & S. (1998). An antimicrobial activity of cytolytic t cells mediated by granulysin. Science, 282(5386), 121-125.

[2] Ma, J. , Lu, J. , Huang, H. , Teng, X. , Tian, M. , & Yu, Q. , et al. (2015). Inhalation of recombinant adenovirus expressing granulysin protects mice infected with mycobacterium tuberculosis. Gene Therapy.

Sequence and Features