Difference between revisions of "Part:BBa K2976014"

Revision as of 15:52, 21 October 2019

NF-κB induced promoter-granulysin

NF-κB induced granulyin expression

Usage

2019 CPU-CHINA team designed a pathway that responses to the signal transducted by Toll like receptors, once the protein at the downstream of the pathway, NF-κB, senses the signal, it will be activated and enter into the nucleus, further regulating the specific gene sequence. Here, we choose an NF-κB induced promoter, together with a coding sequence of granulysin, to enable the cells to excrete granulysin outside the cell. Granulysin is a kind of protein that aims at bacteria and lyses them. This part was designed for eliminating the extracellular M. tuberculosis.

Biology

After being activated with Mtb, the activation cluster TLR1:TLR2:CD14 triggers NF-κB signaling pathways via MYD88 and TRAF6. NF-κB proteins exist in the cytoplasm in an inactive form because of their combination with the IκB proteins. IκB proteins mask the nuclear-localization sequences (NLSs) of NF-κB subunits and retain it in the cytoplasm. Activation of TLR1:TLR2:CD14 cluster cause the degradation of IκB proteins by proteasomes. Then, NF-κB subunits could pass through the nuclear pore complex (NPC) and cause the expression of an array of pro-inflammatory cytokines and chemokines or can bind the NF-κB induced promoter and initiate transcription of the downstream gene: granulysin, this protein was excrete extracellularlly, targeting the free Mtb within the tissues, and eliminating them.

Characterization

We transfected the plamid (NF-κB-granulysin) into the RAW264.7 cells, followed with the extinction with Pam3Cys, the agonist, after being cultured for 24 hours, the cells and supernatant were harvested after another incubation of 24 hours. The supernatant was obtained for detection of granulysin with ELISA(Fig.1) and western blot(Fig.2).

Figure 1: Induced secretion of granulysin.

Figure 2: After being stimulated by Pam3Cys, the phosphorylation of IκB increased，indicating the promotion of NF-κB pathway.

The result of ELISA shows that granulysin was excreted outside of the cells, and was detected in the cell culture fluid. Meanwhile, adding the agonist Pam3Cys significantly increases the concentration of granulysin in the fluid. Combined with the result of western blot, the higher phosphorylation level of IκB after stimulation indicated that more NF-κB was released from the cytoplasm, leading to the expression of downstream gene—granulysin.

To confirm that this plasmid indeed works, we constructed a time-killing curve by way of culturing M.smegtitis with cell culture fluid containing granulysin. As is shown in Figure 3, the sterilizing ability of cells transfected with granulysin showed a slight superiority compared with the control group, while the agonist enables a remarkable effect of death among bacteria.

Figure 3: The time-killing Curve of cells transfected with granulysin and granulysin with Pam3Cys stimulation.

Sequence and Features