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                 <p style="margin-top:30px">Our project aims to induce pyroptosis to tumor cells through Salmonella
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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 trigger by infection of Salmonella, 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 shrinkage of the cell which is different from pyroptosis. In order to demonstrate
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                    the generation of pyroptosis is cased by the N-terminal of GSDMD rather than infection of
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                    Salmonella, 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 infect with Δ<i>sifA</i> Salmonella. Inducer ATc (16
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                    μg/mL) were
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                    added after 5 h infection. Microscopy shows that eGFP-GSDMD-N275 locate in host cell cytoplasm
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                    after 5 min of induction and trigger pyroptosis after 30 min of induction (Figure 1). After 1.5 h
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                    of induction, Hela GSDMD KO cells undergo second necrosis cause by bacterial infection without
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                    inducer. Morphology of this process is similar to pyroptosis<sup>1</sup>. Thus, these population of
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                    ruptured
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                    cells were counted. There are 1.96-fold change between control group and induced group (Figure 2).
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                    So ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by
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                    bacterial infection. These results demonstrate that we successfully implement our goal. </p>
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             </div>
 
             </div>
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                     <img src="https://static.igem.org/mediawiki/2018/b/b3/T--HZAU-China--basicPart4.png" width=100% alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/b/b3/T--HZAU-China--basicPart4.png" width=100% alt="">
 
                 </div>
 
                 </div>
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                 <p>Figure 1. Hela GSDMD KO cell line were infected with ΔsifA SL1344 containing high copy number
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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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                    plasmids which express eGFP-GSDMD-N275 under the control of P<sub>tet</sub>. Photos were capture
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                    after 5 min, 30 min, 1.5h of induction.</p>
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                 <div style="width: 40%; margin: 30px auto">
 
                 <div style="width: 40%; margin: 30px auto">
 
                     <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="">
 
                 </div>
 
                 </div>
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                 <p>Figure 2. Ruptured cells in a field of view were counted (n=8). (Click here to see the method)</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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                <div class="collapseDiv">
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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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                    <div id="zhedie3" class="text-success text-left">
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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>
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                </div>>
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            <div id="float03">
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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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                </p>
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<h3>★  ALERT! </h3>
 
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
 
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
 
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<h1>Demonstrate</h1>
 
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<h3>Gold Medal Criterion #4</h3>
 
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<p>
 
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
 
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</p>
 
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<p>
 
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
 
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</p>
 
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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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