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

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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 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 contents¹.

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

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 (Figure 1). We also tested the cell viability though an ATP assay (CellTiter-Glo^® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (Figure 2).

Figure 1.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.

Method

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. 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).

Method

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) in Salmonella enterica serovar Typhimurium str. SL1344 ΔsifA is under the control of Ptet. 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. CFU comparison between the SL1344 ΔsifA 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).

Method

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) is under the control of tet promoter in ΔsifA SL1344. Hela GSDMD KO cells were infected with ΔsifA 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 (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, the population of ruptured cells was counted. There are 2-fold change between control group and induced group (Figure 5.). So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial infection.

Figure 4.. Hela GSDMD KO cells were infected with ΔsifA 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.

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

Method

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

@@ Line 796: / Line 796: @@
                  <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>
                  <div class="h2">The N-terminal of GSDMD execute the function of pyroptosis in cells</div>
-                 <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® 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 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>
                  <div style="width: 40%; margin: 30px auto">
-                     <img src="https://static.igem.org/mediawiki/2018/3/38/T--HZAU-China--interlab01.png" width=100% alt="">
+                     <img src="https://static.igem.org/mediawiki/2018/3/38/T--HZAU-China--interlab01.png" width="100%" alt="">
                  </div>
-                 <p>Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into
+                 <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>
-                    Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.</p>
                  <div class="collapseDiv">
                      <label for="zhedie-toggle1">Method</label>
-                     <input type="checkbox" id="zhedie-toggle1" />
+                     <input type="checkbox" id="zhedie-toggle1">
                      <div id="zhedie1" class="text-success text-left">
                          <b>Preparation of Cells for transfection</b><br>
@@ Line 811: / Line 810: @@
 . Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow.<br>
                          <b>Transfection</b><br>
-. Dilute 0.5 μg DNA into 50 μl jetPRIME® buffer (supplied). Mix by vortexing.<br>
+. Dilute 0.5 μg DNA into 50 μl jetPRIME<sup>®</sup> buffer (supplied). Mix by vortexing.<br>
-. Add 1 μl jetPRIME®, vortex for 10 s, spin down briefly.<br>
+. Add 1 μl jetPRIME<sup>®</sup>, vortex for 10 s, spin down briefly.<br>
 . Incubate for 10 min at RT.<br>
 . Add 50μl of transfection mix per well drop wise onto the cells in serum containing
@@ Line 823: / Line 822: @@
                  </div>
                  <div style="width: 50%; margin: 30px auto">
-                     <img src="https://static.igem.org/mediawiki/2018/f/f5/T--HZAU-China--basicPart2.png" width=100% alt="">
+                     <img src="https://static.igem.org/mediawiki/2018/f/f5/T--HZAU-China--basicPart2.png" width="100%" alt="">
                  </div>
-                 <p>Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373,
+                 <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>
-                    pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was
-                    measured (n=6).</p>
                  <div class="collapseDiv">
                      <label for="zhedie-toggle2">Method</label>
-                     <input type="checkbox" id="zhedie-toggle2" />
+                     <input type="checkbox" id="zhedie-toggle2">
                      <div id="zhedie2" class="text-success text-left">
                          <b>Preparation of Cells for Infection</b><br>
@@ Line 858: / Line 855: @@
                  </div>
                  <div class="h2">The N-terminal of GSDMD lyses bacteria</div>
-                 <p>Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of P<sub>tet</sub>
+                 <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>
-                    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>
                  <div style="width: 30%; margin: 30px auto">
-                     <img src="https://static.igem.org/mediawiki/2018/f/fb/T--HZAU-China--basicPart3.jpg" width=100% alt="">
+                     <img src="https://static.igem.org/mediawiki/2018/f/fb/T--HZAU-China--basicPart3.jpg" width="100%" alt="">
                  </div>
-                 <p>Figure 3. In each group, ATc (16μg/ml) was added into medium when bacteria grown to logarithmic
+                 <p>
-                    phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only
+    <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>
-                    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>
                  <div class="collapseDiv">
                      <label for="zhedie-toggle3">Method</label>
-                     <input type="checkbox" id="zhedie-toggle3" />
+                     <input type="checkbox" id="zhedie-toggle3">
                      <div id="zhedie3" class="text-success text-left">
 . Cell are cultured overnight in LB broth containing corresponding antibiotics, and
@@ Line 884: / Line 876: @@
                  </div>
                  <div class="h2">The N-terminal of GSDMD from lytic bacteria induce cell pyroptosis.</div>
-                 <p>Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of Tet
+                 <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>
-                    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>
                  <div style="width: 90%; margin: 0 auto">
-                     <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>
-                 <p>Figure 4. Hela GSDMD KO cell line were infected with ΔsifA SL1344 containing high copy number
+                 <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>
-                    plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were captured after 5 min
-                    of induction. </p>
                  <div style="width: 50%; margin: 0 auto">
-                     <img src="https://static.igem.org/mediawiki/2018/2/22/T--HZAU-China--basicPart5.png" width=100% alt="">
+                     <img src="https://static.igem.org/mediawiki/2018/2/22/T--HZAU-China--basicPart5.png" width="100%" alt="">
                  </div>
                  <p>Figure 5. Ruptured cells in a field of view were counted.</p>
                  <div class="collapseDiv">
                      <label for="zhedie-toggle4">Method</label>
-                     <input type="checkbox" id="zhedie-toggle4" />
+                     <input type="checkbox" id="zhedie-toggle4">
                      <div id="zhedie4" class="text-success text-left">
                          <b>Preparation of Cells for Infection</b><br>
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                      <table class="table table-bordered table-hover">
                          <thead>
-                             <th>Name</th>
+                             <tr><th>Name</th>
                              <th>Type</th>
                              <th>Description</th>