Difference between revisions of "Part:BBa K2632003"
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Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that N-terminal of Gasdermin D acts as a effector of pyroptosis. Full length Gasdermin D is cleaved by Caspase 1 then release the PFD(pore-forming domain) which can oligomerize on the cell membrane. Formation of pore causes cell swelling, rupture of the membrane and massive leakage of cytosolic contents.
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Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that N-terminal of Gasdermin D acts as a effector of pyroptosis. Full length Gasdermin D is cleaved by Caspase 1 then release the PFD(pore-forming domain) which can oligomerize on the cell membrane. Formation of pore causes cell swelling, rupture of the membrane and massive leakage of cytosolic contents<sup>1</sup>.
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We respectively fused EGFP with GSDMD-N275, GSDMD FL(full length) and L290D,Y373D,A377D mutation of GSDMD FL. Then transfect these plasmids into HEK293T cell. Microscopy of GSDMD-N275 undergoing pyroptosis, but GSDMD full length did not induce pyroptosis(Fig 1). We also test the cell viability though an ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrate that GSDMD-N275 and mutation of GSDMD FL have different ability to induce pyroptosis(Fig 2).
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We respectively fused eGFP with GSDMD-N275, GSDMD FL (full length) and L290D, Y373D, A377D mutants of GSDMD FL. Then these plasmids were transfected into Hela GSDMD KO cell. Microscopy of cells transfecting GSDMD-N275 undergoing pyroptosis, but GSDMD full length did not induce pyroptosis (Fig.1). We also test the cell viability though an ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (Fig.2).  
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<img src="https://static.igem.org/mediawiki/2018/9/90/T--HZAU-China--pCS2-EGFP-GSDMD-FL.png" width="400px"/>
 
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Figure 1. pCS2-EGFP-GSDMD FL（left）, pCS2-EGFP-GSDMD-N275（right） were transfected respectively into 293T cells.  
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Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.  
 
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Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373, pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was measured (n=6). (Click here to see method)
 
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<img src="https://static.igem.org/mediawiki/2018/0/0f/T--HZAU-China--GSDMD-mutation-viability.png" width="700px"/>
 
<img src="https://static.igem.org/mediawiki/2018/0/0f/T--HZAU-China--GSDMD-mutation-viability.png" width="700px"/>
 
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Figure 2. pCS2-EGFP-GSDMD FL, pCS2-EGFP-GSDMD-N275, pCS2-EGFP-GSDMD L290D, pCS2-EGFP-GSDMD Y373, pCS2-EGFP-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was measured.
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Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in <i>ΔsifA</i> SL1344. Hela GSDMD KO cell line were infect with <i>ΔsifA</i> SL1344. Inducer ATc (16μg/mL) were added after 3h infetion. Microscopy shows that eGFP-GSDMD-N275 locate in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Fig.3).After 1.5 h of induction, Hela GSDMD KO cells undergo second necrosis cause by bacterial infection without inducer. Morphology of this process is similar to pyroptosis<sup>2</sup>. Thus, these population of ruptured cells were counted. There are two fold change between control group and induced group (Fig.4). So  ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by bacterial infection.
 
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Figure 3. Hela GSDMD KO cell line were infected with <i>ΔsifA</i> SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were capture after 5 min of induction.
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Figure 4. Ruptured cells in a field of view were counted.
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Revision as of 07:58, 13 October 2018


N-terminal of GasderminD (1-275aa)

Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that N-terminal of Gasdermin D acts as a effector of pyroptosis. Full length Gasdermin D is cleaved by Caspase 1 then release the PFD(pore-forming domain) which can oligomerize on the cell membrane. Formation of pore causes cell swelling, rupture of the membrane and massive leakage of cytosolic contents1. We respectively fused eGFP with GSDMD-N275, GSDMD FL (full length) and L290D, Y373D, A377D mutants of GSDMD FL. Then these plasmids were transfected into Hela GSDMD KO cell. Microscopy of cells transfecting GSDMD-N275 undergoing pyroptosis, but GSDMD full length did not induce pyroptosis (Fig.1). We also test the cell viability though an ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (Fig.2).

Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.


Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373, pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was measured (n=6). (Click here to see method)

Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in ΔsifA SL1344. Hela GSDMD KO cell line were infect with ΔsifA SL1344. Inducer ATc (16μg/mL) were added after 3h infetion. Microscopy shows that eGFP-GSDMD-N275 locate in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Fig.3).After 1.5 h of induction, Hela GSDMD KO cells undergo second necrosis cause by bacterial infection without inducer. Morphology of this process is similar to pyroptosis2. Thus, these population of ruptured cells were counted. There are two fold change between control group and induced group (Fig.4). So ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by bacterial infection.


Figure 3. Hela GSDMD KO cell line were infected with ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were capture after 5 min of induction.
Figure 4. Ruptured cells in a field of view were counted.

Reference

Park S H, Zheng J H, Nguyen V H, et al. RGD Peptide cell-surface display enhances the targeting and therapeutic efficacy of attenuated salmonella-mediated cancer therapy[J]. Theranostics, 2016, 

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]