Difference between revisions of "Team:HZAU-China/Demonstrate"

 
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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>
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                     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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                     Salmonella. Hela GSDMD KO (knockout) 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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                     <img src="https://static.igem.org/mediawiki/2018/1/16/T--HZAU-China--Demonstrate2.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/1/16/T--HZAU-China--Demonstrate2.png" width=100% alt="">
 
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                 <p><b>Figure 2.</b> Ruptured cells in a field of view were counted (n=8).</p>
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                 <p><b>Figure 2.</b> Ruptured cells in a field of view were counted (n=8).</p><br>  
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                <p>In these experiments, the choice of MOI (multiplicity of infection) and infection time were directed by <a href="https://2018.igem.org/Team:HZAU-China/Model">Modeling</a>.</p>          
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                    <label for="zhedie-toggle3">Method</label>
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                        <b>Preparation of Cells for Infection</b><br>
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                        1. Grow Hela GSDMD KO cells in a humidified 37℃, 5% CO<sub>2</sub> tissue-culture incubator.<br>
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                        2. Count the cells using a hemocytometer. Seed in 24-well (5×10^4 per well) and grow
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                        overnight.<br>
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                        <b>Preparation of Bacteria</b><br>
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                        1. Grow <i>Salmonella</i> bacterial cells overnight (16 h) in 2 mL LB in a 15-mL tube. Incubate at 37℃ in a shaking
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                        incubator (200 rpm).<br>
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                        2. Subculture <i>Salmonella</i> bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a
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                        loosely capped 50-mL tube. Incubate at 37℃ in a shaking incubator (200 rpm) to late log
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                        phase.<br>
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                        3. Pellet 1 mL of the <i>Salmonella</i> bacterial cells subculture by centrifugation at 1,000×g in a microfuge for 2
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                        min at room temperature.<br>
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                        4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
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                        <b>Infection</b><br>
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                        1. Aspirate media and rinse the monolayer twice with PBS.<br>
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                        2. Inoculate cells with bacteria cells (MOI = 100) by adding bacteria cells directly to the cell-culture
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                        supernatant.<br>
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                        3. Incubate for 2 h at 37℃ in 5% CO<sub>2</sub>.<br>
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                        4. Aspirate media and wash<br>
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                        5. Add fresh Grow Meduim (GM) containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37℃ in 5% CO<sub>2</sub> for 2
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                        h.<br>
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                        6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.<br>
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                        7.Add 16 μg/mL ATc for remainder of experiment.<br>
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                        Observation is taken after 5 min, 30 min, 90 min.<br><br>
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Latest revision as of 02:41, 18 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 (knockout) 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!!!

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.

Figure 2. Ruptured cells in a field of view were counted (n=8).


In these experiments, the choice of MOI (multiplicity of infection) and infection time were directed by Modeling.

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