Difference between revisions of "Team:HZAU-China/Basic Part"

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                 <div class="h1">N-terminal of Gasdermin D (1-275aa)</div>
 
                 <div class="h1">N-terminal of Gasdermin D (1-275aa)</div>
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                 <p>Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that the N-terminal of GSDMD acts as an effector of pyroptosis. Full length GSDMD (GSDMD FL) is cleaved by Caspase 1, releasing the pore-forming domain (GSDMD-N275) which can oligomerize and make pores on the cell membrane. Formation of pores causes cell to swell, leading to membrane rupture and massive leakage of cytosolic contents<sup>1</sup>.</p>
+
                 <p>Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that
 +
                    the N-terminal of Gasdermin D (pore-forming domain) acts as an effector of pyroptosis. Full length
 +
                    Gasdermin D is cleaved by Caspase 1 then release the PFD (pore-forming domain) which can
 +
                    oligomerize on the cell membrane. Formation of pores causes cell swelling, rupturing of the
 +
                    membrane and the massive leakage of cytosolic contents<sup>1</sup>.</p>
 
                 <div class="h2">The N-terminal of GSDMD execute the function of pyroptosis in cells</div>
 
                 <div class="h2">The N-terminal of GSDMD execute the function of pyroptosis in cells</div>
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                 <p>We fused eGFP with GSDMD-N275 and GSDMD FL (full length), respectively. Then the corresponding plasmids were transfected into Hela GSDMD KO cell. Cell microscopy showed that the cells transfected with GSDMD-N275 underwent pyroptosis while the cells with GSDMD FL did not (<b>Figure 1</b>). We also tested the cell viability though an ATP assay (CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (<b>Figure 2</b>).</p>
+
                 <p>We respectively fused eGFP with GSDMD-N275 and GSDMD FL (full length). Then these plasmids were
 +
                    transfected into Hela GSDMD KO cell. Microscopy of cells transfecting GSDMD-N275 undergoing
 +
                    pyroptosis, but GSDMD full length did not induce pyroptosis (Figure 1). We also tested the cell
 +
                    viability through ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that
 +
                    GSDMD-N275 and mutants of GSDMD FL had different ability to induce pyroptosis (Figure 2).</p>
 
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                 <p><b>Figure 1.</b>Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL and pCS2-eGFP-GSDMD-N275, respectively. Pyroptotic cells are pointed by red arrow.</p>
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                 <p>Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into
 +
                    Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.</p>
 
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                         <b>Preparation of Cells for transfection</b><br>
 
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                         2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow.<br>
 
                         2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow.<br>
 
                         <b>Transfection</b><br>
 
                         <b>Transfection</b><br>
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                         1. Dilute 0.5 μg DNA into 50 μl jetPRIME<sup>®</sup> buffer (supplied). Mix by vortexing.<br>
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                         1. Dilute 0.5 μg DNA into 50 μl jetPRIME® buffer (supplied). Mix by vortexing.<br>
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                         2. Add 1 μl jetPRIME<sup>®</sup>, vortex for 10 s, spin down briefly.<br>
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                         2. Add 1 μl jetPRIME®, vortex for 10 s, spin down briefly.<br>
 
                         3. Incubate for 10 min at RT.<br>
 
                         3. Incubate for 10 min at RT.<br>
 
                         4. Add 50μl of transfection mix per well drop wise onto the cells in serum containing
 
                         4. Add 50μl of transfection mix per well drop wise onto the cells in serum containing
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                 <p><b>Figure 2</b>. Cell viability of the 293T cells transfected with pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373D, pCS2-Flag-GSDMD A377D, respectively. Asterisks indicate the statistically significant differences. ATP-based cell viability was measured (n=6).</p>
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                 <p>Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373,
 +
                    pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was
 +
                    measured (n=6).</p>
 
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                         <b>Preparation of Cells for Infection</b><br>
 
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                 <div class="h2">The N-terminal of GSDMD lyses bacteria</div>
 
                 <div class="h2">The N-terminal of GSDMD lyses bacteria</div>
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                 <p>Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) in <i>Salmonella enterica</i> serovar Typhimurium str. SL1344 <i>ΔsifA</i> is under the control of P<i>tet</i>. The colony-forming unit (CFU) was measured for counting the number of viable bacteria (<b>Figure 3.</b>). This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.</p>
+
                 <p>Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of P<sub>tet</sub>
 +
                    in Δ<i>sifA</i> SL1344.
 +
                    The colony-forming unit (CFU) was measured for counting the number of viable bacteria (Figure 3).
 +
                    This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.</p>
 
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                 <p>Figure 3. In each group, ATc (16μg/ml) was added into medium when bacteria grown to logarithmic
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    <b>Figure 3.</b> CFU comparison between the SL1344 <i>ΔsifA</i> cells with eGFP-GSDMD-N275 plasmid and with the empty vector. In each group, ATc (15μg/ml) was added into medium when bacteria grown to logarithmic phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only express TetR under the control of P_tet . Bacterial colony-forming units (CFU) for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3). </p>
+
                    phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only
 +
                    expresses TetR under the control of P<sub>tet</sub>. Bacterial colony-forming units (CFU) for
 +
                    vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3). </p>
 
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                         1. Cell are cultured overnight in LB broth containing corresponding antibiotics, and
 
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                 <div class="h2">The N-terminal of GSDMD from lytic bacteria induce cell pyroptosis.</div>
 
                 <div class="h2">The N-terminal of GSDMD from lytic bacteria induce cell pyroptosis.</div>
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                 <p>Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in <i>ΔsifA</i> SL1344. Hela GSDMD KO cells were infected with <i>ΔsifA</i> SL1344. Inducer ATc (15μg/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 4.</b>). After 1.5 h of induction, Hela GSDMD KO cells undergo second necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis2. Thus, the population of ruptured cells was counted. There are 2-fold change between control group and induced group (<b>Figure 5.</b>). So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial infection.</p>
+
                 <p>Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of Tet
 +
                    promoter in Δ<i>sifA</i> SL1344. Hela GSDMD KO cell line were infected with ΔsifA SL1344. Inducer
 +
                    ATc
 +
                    (16μg/mL) were added after 3 h infection. Microscopy showed that eGFP-GSDMD-N275 located in
 +
                    cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Figure 4).
 +
                    After 1.5 h of induction, Hela GSDMD KO cells undergo second necrosis caused by bacterial infection
 +
                    without inducer. Morphology of this process is similar to pyroptosis<sup>2</sup>. Thus, these
 +
                    population of
 +
                    ruptured cells were counted. There are two folds change between control group and induced group
 +
                    (Figure 5). So ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not
 +
                    by bacterial infection.</p>
 
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                 <p><b>Figure 4.</b>. Hela GSDMD KO cells were infected with <i>ΔsifA</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>
+
                 <p>Figure 4. Hela GSDMD KO cell line were infected with ΔsifA SL1344 containing high copy number
 +
                    plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were captured after 5 min
 +
                    of induction. </p>
 
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                 <p>Figure 5. Ruptured cells in a field of view were counted.</p>
 
                 <p>Figure 5. Ruptured cells in a field of view were counted.</p>
 
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Revision as of 13:43, 16 October 2018

N-terminal of Gasdermin D (1-275aa)

Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that the N-terminal of Gasdermin D (pore-forming domain) acts as an effector of pyroptosis. Full length Gasdermin D is cleaved by Caspase 1 then release the PFD (pore-forming domain) which can oligomerize on the cell membrane. Formation of pores causes cell swelling, rupturing of the membrane and the massive leakage of cytosolic contents1.

The N-terminal of GSDMD execute the function of pyroptosis in cells

We respectively fused eGFP with GSDMD-N275 and GSDMD FL (full length). Then these plasmids were transfected into Hela GSDMD KO cell. Microscopy of cells transfecting GSDMD-N275 undergoing pyroptosis, but GSDMD full length did not induce pyroptosis (Figure 1). We also tested the cell viability through ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL had different ability to induce pyroptosis (Figure 2).

Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.

Preparation of Cells for transfection
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow.
Transfection
1. Dilute 0.5 μg DNA into 50 μl jetPRIME® buffer (supplied). Mix by vortexing.
2. Add 1 μl jetPRIME®, vortex for 10 s, spin down briefly.
3. Incubate for 10 min at RT.
4. Add 50μl of transfection mix per well drop wise onto the cells in serum containing medium, and distribute evenly.
5. Gently rock the plates back and forth and from side to side.
6. If needed, replace transfection medium after 4 h by cell growth medium and return the plates to the incubator.
Observation is taken after 1.5 h

Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373, pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was measured (n=6).

Preparation of Cells for Infection
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (9× 10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 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 (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 3 h at 37 °C in 5% CO2.
4. Aspirate media and rinse the monolayer twice with PBS.
5. Add fresh GM containing 100 μg/mL gentamicin and incubate at 37 °C in 5% CO2.
Observation is taken after 2 h.
The N-terminal of GSDMD lyses bacteria

Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of Ptet in ΔsifA SL1344. The colony-forming unit (CFU) was measured for counting the number of viable bacteria (Figure 3). This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.

Figure 3. In each group, ATc (16μg/ml) was added into medium when bacteria grown to logarithmic phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only expresses TetR under the control of Ptet. Bacterial colony-forming units (CFU) for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3).

1. Cell are cultured overnight in LB broth containing corresponding antibiotics, and dilute each 1 volume overnight cultures with 100 volume fresh LB containing antibiotics. Culture in 37℃ 200 rpm.
2. When OD reaching to 0.6-0.8, add anhydrotetracycline with final concentration of μg/ml to induce the expression of EGFP-GSDMD-N275.
3. Take 100 μl diluted culture to plate on LB agar plates containing appropriate concentration of antibody after 1.5 hours of induce.
Observation is taken overnight.
The N-terminal of GSDMD from lytic bacteria induce cell pyroptosis.

Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of Tet promoter in ΔsifA SL1344. Hela GSDMD KO cell line were infected with ΔsifA SL1344. Inducer ATc (16μg/mL) were added after 3 h infection. Microscopy showed that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Figure 4). After 1.5 h of induction, Hela GSDMD KO cells undergo second necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis2. Thus, these population of ruptured cells were counted. There are two folds change between control group and induced group (Figure 5). So ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by bacterial infection.

Figure 4. Hela GSDMD KO cell line were infected with ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were captured after 5 min of induction.

Figure 5. Ruptured cells in a field of view were counted.

Preparation of Cells for Infection
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 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 bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 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 (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 2 h at 37 °C in 5% CO2.
4. Aspirate media and wash.
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2.
Observation is taken after 5 min, 30 min, 1.5 h.
Basic Part Table
Name Type Description Designer Length(bp)
BBa_K2632002 Promoter Promoter sifA Mo Qiqin 331
BBa_K2632003 Coding N-terminal of GasderminD (1-275aa) Zhujun Xia 825
BBa_K2632004 Coding Full length Gasdermin D Zhujun Xia 1455
BBa_K2632005 Coding L290D mutant of full length Gasdermin D Zhujun Xia 1455
BBa_K2632007 Coding A377D mutant of full length Gasdermin D. Zhujun Xia 1455
Reference

1 Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111-116, doi:10.1038/nature18590 (2016).

2 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).

Basic Part

N-terminal of Gasdermin D

Basic Part Table

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Composite Part