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

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         <div class="zhengwen">
 
             <div id="float01" class="cur">
 
             <div id="float01" class="cur">
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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 aims is to induce pyroptosis in tumor cells through  
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                     translocate a pyroptosis
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                     translocating a pyroptosis
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                     associate protein GSDMD. In fact, pyroptosis also can be triggered by infection of <i>Salmonella</i>, but
+
                     associate protein GSDMD by <i>Salmonella</i>. In fact, pyroptosis also can be triggered by the infection of <i>Salmonella</i>, but
−
                     Hela GSDMD KO cell line undergoing apoptosis after infection. In morphology, apoptosis is
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                     Hela GSDMD KO cell line undergoes apoptosis after infection. In morphology, apoptosis is
−
                     characterised by the shrinkage of the cell which is different from pyroptosis. In order to demonstrate
+
                     characterized 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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                     pyroptosis is caused by the N-terminal of GSDMD (GSDMD-N275) rather than the infection of
−
                     <i>Salmonella</i>, Hela GSDMD KO cell line was used in our experiment. Expression of the N-terminal of
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                     <i>Salmonella</i> itself, 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>
+
                     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> SL1344. Inducer ATc (16μg/mL) were added 3 h 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 underwent 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>
+
                     Salmonella. Hela GSDMD KO cell line were infected with Δ<i>sifA</i> SL1344. Inducer ATc (16μg/mL) were added 3 h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and triggering pyroptosis after 30 min of induction (<b>Figure 1</b>). After 1.5 h of induction, Hela GSDMD KO cells underwent the 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 are 2-fold changes 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>
  
 
             </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><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, 30min, and 1.5h after induction, respectively. </p>
 
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                 <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="">
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                         <b>Preparation of Cells for Infection</b><br>
 
                         <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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                         1. Grow Hela GSDMD KO cells in a humidified 37℃, 5% CO2 tissue-culture incubator.<br>
−
                         2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow
+
                         2. Count the cells using a hemocytometer. Seed in 24-well (5×10^4 per well) and grow
 
                         overnight.<br>
 
                         overnight.<br>
 
                         <b>Preparation of Bacteria</b><br>
 
                         <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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                         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
 
                         incubator (200 rpm).<br>
 
                         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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                         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 °C in a shaking incubator (200 rpm) to late log
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                         loosely capped 50-mL tube. Incubate at 37℃ in a shaking incubator (200 rpm) to late log
 
                         phase.<br>
 
                         phase.<br>
−
                         3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 g in a microfuge for 2
+
                         3. Pellet 1 mL of the <i>Salmonella</i> bacterial cells subculture by centrifugation at 1,000×g in a microfuge for 2
 
                         min at room temperature.<br>
 
                         min at room temperature.<br>
 
                         4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
 
                         4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
 
                         <b>Infection</b><br>
 
                         <b>Infection</b><br>
 
                         1. Aspirate media and rinse the monolayer twice with PBS.<br>
 
                         1. Aspirate media and rinse the monolayer twice with PBS.<br>
−
                         2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture
+
                         2. Inoculate cells with bacteria cells (MOI = 100) by adding bacteria cells directly to the cell-culture
 
                         supernatant.<br>
 
                         supernatant.<br>
−
                         3. Incubate for 2 h at 37 °C in 5% CO2.<br>
+
                         3. Incubate for 2 h at 37℃ in 5% CO2.<br>
 
                         4. Aspirate media and wash<br>
 
                         4. Aspirate media and wash<br>
−
                         5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2 for 2
+
                         5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37℃ in 5% CO2 for 2
 
                         h.<br>
 
                         h.<br>
 
                         6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.<br>
 
                         6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.<br>

Revision as of 16:04, 17 October 2018

Our project aims is to induce pyroptosis in tumor cells through translocating a pyroptosis associate protein GSDMD by Salmonella. In fact, pyroptosis also can be triggered by the infection of Salmonella, but Hela GSDMD KO cell line undergoes apoptosis after infection. In morphology, apoptosis is characterized by the shrinkage of the cell, which is different from pyroptosis. In order to demonstrate pyroptosis is caused by the N-terminal of GSDMD (GSDMD-N275) rather than the infection of Salmonella itself, Hela GSDMD KO cell line was used in our experiment. 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 SL1344. Inducer ATc (16μg/mL) were added 3 h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and triggering pyroptosis after 30 min of induction (Figure 1). After 1.5 h of induction, Hela GSDMD KO cells underwent the secondary necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis1. Thus, the population of ruptured cells was counted. There are 2-fold changes between control group and induced group (Figure 2). 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.

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, 30min, and 1.5h after induction, respectively.

Figure 2. Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of view were counted.

Preparation of Cells for Infection
1. Grow Hela GSDMD KO cells in a humidified 37℃, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5×10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow Salmonella bacterial cells overnight (16 h) in 2 mL LB in a 15-mL tube. Incubate at 37℃ in a shaking incubator (200 rpm).
2. Subculture Salmonella bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37℃ in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella bacterial cells subculture by centrifugation at 1,000×g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacteria cells (MOI = 100) by adding bacteria cells directly to the cell-culture supernatant.
3. Incubate for 2 h at 37℃ in 5% CO2.
4. Aspirate media and wash
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37℃ in 5% CO2 for 2 h.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.
7.Add 16 μg/mL ATc for remainder of experiment.
Observation is taken after 5 min, 30 min, 1.5 h.

Reference

1 He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1beta secretion. Cell research 25, 1285-1298, doi:10.1038/cr.2015.139 (2015).

Description Design Results
Demonstrate
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