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

 
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                 <p>Our project aims to induce pyroptosis to tumor cells through <i>Salmonella</i>
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                 <p>Our project goal is to induce pyroptosis in tumor cells through  
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                     translocate a pyroptosis
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                     translocating a pyroptosis-associated protein GSDMD by <i>Salmonella</i>. Expression of the N-terminal of
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                    associate protein GSDMD. In fact, pyroptosis also can be triggered by infection of <i>Salmonella</i>, but
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                     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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                    Hela GSDMD KO cell line undergoing apoptosis after infection. In morphology, apoptosis is
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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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                    characterised by the shrinkage of the cell which is different from pyroptosis. In order to demonstrate
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                    the generation of pyroptosis is caused by the N-terminal of GSDMD (GSDMD-N275) rather than infection of
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                    <i>Salmonella</i>, Hela GSDMD KO cell line was used in our experiment. Expression of the N-terminal of
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                     GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of 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> SL1344. Inducer ATc (16μg/mL) were added 3h after infection. Microscopy shows that eGFP-GSDMD-N275 locates in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (<b>Figure 1</b>). After 1.5 h of induction, Hela GSDMD KO cells undergo secondary necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis<sup>1</sup>. Thus, the population of ruptured cells was counted. There is 2-fold change between control group and induced group (<b>Figure 2</b>). So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial infection. These results demonstrate that we successfully implement our goal. </p>
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                     <img src="https://static.igem.org/mediawiki/2018/b/b3/T--HZAU-China--basicPart4.png" width=100% alt="">
 
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                 <p><b>Figure 1.</b>  Hela GSDMD KO cells were infected with Δ<i>sifA</i> SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were captured 5 min, 30min, 1.5h after induction, respectively. </p>
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                 <p><b>Figure 1.</b>  Hela GSDMD KO cells were infected by <i>Salmonella</i> Δ<i>sifA</i> 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. </p>
 
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                     <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> Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of view were counted.</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 °C, 5% CO2 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 bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
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                        incubator (200 rpm).<br>
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                        2. Subculture bacteria 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 °C 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 Salmonella subculture by centrifugation at 1000 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 (MOI = 100) by adding bacteria directly to the cell-culture
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                        supernatant.<br>
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                        3. Incubate for 2 h at 37 °C in 5% CO2.<br>
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                        4. Aspirate media and wash<br>
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                        5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2 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, 1.5 h.<br><br>
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                <div class="h1">Reference</div>
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                <p>1 He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1beta
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                    secretion. Cell research 25, 1285-1298, doi:10.1038/cr.2015.139 (2015).
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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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