Difference between revisions of "Part:BBa K2976009"

Revision as of 16:29, 24 October 2020

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 Mtb 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.

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.

MIT_MAHE 2020

Summary

Nuclear factor-κB (NF-κB) consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival. 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 Mtb infected macrophages. It indirectly leads to production of cytokines, chemokines and NO.

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.

Added by KEYSTONE_A 2020

NF-κB reponsive promoter constructions with different NF-κB-box repeat numbers (4× or 5×) and different promoter cores (PTAL and PMIN) were characterized. TNFα and IL-1β are two inflammatory factors that can activate NF-κB pathway as input. Luciferase activity was measured as output. Fold induction (FI) is defined by ON-OFF ratio. Promoter with 5× NF-κB-box and PMIN core shows best responsiveness and lowest leaky expression.

Figure 3.Optimization of NF-κB responsive promoter by increasing NF-κB-box repeat number and lowering promoter strength

Reference:

Smole, A., Lainšček, D., Bezeljak, U., Horvat, S., & Jerala, R. (2017). A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular therapy, 25(1), 102-119.

Sequence and Features