Difference between revisions of "Part:BBa K2976014"

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===Characterization===
 
===Characterization===
 
<p>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).  
 
<p>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).  
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[[File:T--CPUCHINA--GRN.jpg|380px|thumb|left|Figure 1: Induced secretion of granulysin.]]
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[[File:T--CPUCHINA--GRN.png|380px|thumb|left|Figure 1: Induced secretion of granulysin.]]
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[[File:T--CPUCHINA--PIKB.jpg|380px|thumb|left|Figure 2: After being stimulated by Pam3Cys, the phosphorylation of IκB increased，indicating the promotion of NF-κB pathway.]]
 
[[File:T--CPUCHINA--PIKB.jpg|380px|thumb|left|Figure 2: After being stimulated by Pam3Cys, the phosphorylation of IκB increased，indicating the promotion of NF-κB pathway.]]
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Revision as of 15:30, 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.

</p>

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

</p>



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 585
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]