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translocating a pyroptosis-associated protein GSDMD by <i>Salmonella</i>. Expression of the N-terminal of | translocating a pyroptosis-associated protein GSDMD by <i>Salmonella</i>. Expression of the N-terminal of | ||
GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of the promoter P<sub>tet</sub> in Δ<i>sifA</i> | GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of the promoter P<sub>tet</sub> in Δ<i>sifA</i> | ||
− | Salmonella. Hela GSDMD KO cell line were infected with Δ<i>sifA</i> <i>Salmonella</i>. Inducer anhydrotetracycline (ATc) (16μg/mL) was added 3 h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and pyroptosis was triggered after 30 min of induction (<b>Figure 1</b>). By counting the population of ruptured cells, a 1.96 fold-change was demonstrated between the induced group and the control group (<b>Figure 2</b>). So, the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 as expected. These results demonstrate that we successfully achieved the goal of our project | + | Salmonella. Hela GSDMD KO cell line were infected with Δ<i>sifA</i> <i>Salmonella</i>. Inducer anhydrotetracycline (ATc) (16μg/mL) was added 3 h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and pyroptosis was triggered after 30 min of induction (<b>Figure 1</b>). By counting the population of ruptured cells, a 1.96 fold-change was demonstrated between the induced group and the control group (<b>Figure 2</b>). So, the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 as expected. These results demonstrate that we successfully achieved the goal of our project!!! </p> |
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Revision as of 21:51, 17 October 2018
Our project goal is to induce pyroptosis in tumor cells through translocating a pyroptosis-associated protein GSDMD by Salmonella. Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of the promoter Ptet in ΔsifA Salmonella. Hela GSDMD KO cell line were infected with ΔsifA Salmonella. Inducer anhydrotetracycline (ATc) (16μg/mL) was added 3 h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and pyroptosis was triggered after 30 min of induction (Figure 1). By counting the population of ruptured cells, a 1.96 fold-change was demonstrated between the induced group and the control group (Figure 2). So, the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 as expected. These results demonstrate that we successfully achieved the goal of our project!!!
![](https://static.igem.org/mediawiki/2018/b/b3/T--HZAU-China--basicPart4.png)
Figure 1. Hela GSDMD KO cells were infected by Salmonella ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photographs were captured 5 min, 30 min, and 90 min after induction, respectively.
![](https://static.igem.org/mediawiki/2018/1/16/T--HZAU-China--Demonstrate2.png)
Figure 2. Ruptured cells in a field of view were counted (n=8).