Difference between revisions of "Part:BBa K2976009"

Latest revision as of 10:09, 9 December 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.

Improved by CPU_CHINA 2020

we improved the efficacy of this part. We reconstructed this part into promoter JTi2 consists of five repeats of 5′-AGGGATTTCCC-3′as NF-κB binding site and a minimal CMV promoter. The binding sites repeats is derived from gene vascular cell adhesion molecule 1, VCAM1. The reconstructed promoter exhibits more than two times of efficiency of this original part.

We induce the activation of NF-κB by adding TNF-α in cell culture liquid medium. After inducing for 36h the supernatant of the cell culture was extracted and analysed. We applied SEAP as reporter, the total enzyme activities of SEAP in the supernatant are recorded and evaluated between groups, the enzyme activity of SEAP downstream of JTi2 is more than two times of the original existed part, suggested that the efficiency of reconstructed JTi2 promoter is elevated to more than two times of the existed old part.

Figure 1.promoter efficiency of promoter JTi2

The efficiency of JTi2 promoter can be reflected and analysed by the total enzyme efficiency. The efficiency of reconstructed JTi2 promoter is more than two times of the existed old part BBa_K2976009.

Sequence and Features

Improved by NFLS 2020

Part improvement by NFLS 2020

Team NFLS improved the efficiency of this part by designing a new part called JTi1 consists of five repeats of 5′-GGGTTTCCCC-3′as NF-κB binding site and a minimal CMV promoter. The binding sites repeats is derived from gene vascular cell adhesion molecule 1, VCAM1. This new reconstructed promoter is more than twice as efficient as this original part.

We added a Secreted Alkaline Phosphatase (SEAP) which commonly used as a reporter gene behind the promoter. The enzyme activity of SEAP reflects the efficacy of the promoter.

The improved part is: BBa_K3513006

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The standard curve:

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Sequence and Features