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

Revision as of 17:27, 17 October 2018

Chassis

sifA knock out

SifA maintains the integrity of Salmonella-containing vacuole (SCV) where Salmonella survive and replicate¹. The existence of SCV limits the releasing of GSDMD-N275 into cytoplasm. In addition, the growth of inhibition of ΔsifA mutant in macrophage is remarkable². Thus, we knocked out the sifA gene in order to prevent the stability of SCV and reduce the virulence of Salmonella.

ΔsifA mutant was constructed by using gene editing system based on two-step allelic exchange³.

PCR verification indicated that chromosomal gene sifA was knocked out (Figure 1). Primers named DSIFA F and DSIFA R were used in this PCR (Figure 2).

Figure 1. PCR verification of sifA gene knock out. Lane 1 refers to ΔsifA mutant. Lane 2 refers to WT Salmonella enterica serovar Typhimurium strain SL1344 as a control.

Figure 2. DSIFA F and DSIFA R are the primers in the ORF of sifA. This pair of primers can extend a 436bp product in WT.

Safety

SifA is essential for maintaining vacuolar membrane stability. Cytosolic bacterium can be generated by the function loss of SCV. This population of bacterium was easily recognized and cleaned up by macrophage. Hence, ΔsifA mutant decrease the toxicity to the host. Microscopy demonstrated that ΔsifA mutant was defective for replication in macrophage (Figure 3).

Figure 3. Microscopy of immortalized bone-marrow-derived macrophages (iBMDM) infected with the ΔsifA mutant and WT Salmonella SL1344, respectively. These strains contain high copy number plasmids to express RFP constitutively.

Method

Preparation of Cells for Infection
1. Grow iBMDMs 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 the Bacterial Cells 1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture 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 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella bacteria 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 bacterial cells (MOI = 20) by adding bacterial cells directly to the cell-culture supernatant. Centrifugate at 110 g for 5 min at room temperature.
3. Incubate for 25 min at 37 °C in 5% CO₂.
4. Aspirate media and rinse the monolayer twice with PBS, to remove extracellular bacterial cells.
5. Add fresh GM containing 100 μg/mL gentamicin and incubate for 2 h at 37 °C in 5% CO₂.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for remainder of experiment.
Observation is taken after 11 h.

Targeting

RGD motif can specifically bind to alpha V beta 3 (αVβ3), a well-known biomarker on the surface of tumor cells. We express RGD motif fused with OmpA to surface display it on the outer membrane of bacterial cells. Microscopy shows that bacterial cells expressed Lpp-OmpA-RGD induced by 0.1 mM IPTG can bind to αVβ3-positive MDA-MB-231 cell line. The location of bacterial cells is pointed by red arrow (Figure 4). But cannot bind to αVβ3-negative MCF7 cell line (Figure 5). These results demonstrated that the bacterium gain the function of tumor targeting though display RGD motif.

Figure 4. Microscopy of αVβ3-positive MDA-MB-231 cell line were incubated with E. coli which constructive expressed RFP and inductively expressed RGD motif under the control of P^lac.

Figure 5. Microscopy of αVβ3-negative MCF7 cell line were incubated with E. coli which constructive expressed RFP and inductively expressed RGD motif under the control of lac promoter.

Method

Preparation of Cells
1. Grow MDA-MB-231 and MCF7 in a humidified 37 °C, 5% CO₂ tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (1.5× 10^5 per well) and grow overnight.
Preparation of Bacterial cells 1. Grow bacterial cells overnight 14 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB containing 0.1 mM IPTG in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to early stationary phase.
3. Pellet 1 mL of the bacterial cells 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 bacterial cells (MOI = 100) by adding bacterial cells directly to the cell-culture supernatant.
3. Incubate for 1.5 h at 37 °C in 5% CO₂.
4. Aspirate media and rinse the monolayer three times with PBS.
Observation is taken immediately.

Function of GSDMD

The N-terminal of GSDMD performs the function of cell pyroptosis

We fused eGFP with GSDMD-N275 (N-terminal 275 amino acid) 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 6). We also tested the cell viability through an ATP assay (CellTiter-Glo^® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different abilities to induce pyroptosis (Figure 7).

Figure 6. Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL (above) and pCS2-eGFP-GSDMD-N275 (left), 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 7. 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 ATP assay
1. Grow HEK293T cells in a humidified 37 °C, 5% CO₂ tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 96-well (1 × 10^4 per well) and grow overnight.
3. Transfect 0.5 μg DNA per well.
4. Equilibrate the plate and its contents at room temperature for approximately 30 minutes after 20 h.
5. Add a volume of CellTiter-Glo^® Reagent equal to the volume of cell culture medium present in each well. (add 100 μl of reagent to 100 μl of medium containing cells for a 96-well plate).
6. Mix contents for 2 minutes on an orbital shaker to induce cell lysis.
7. Allow the plate to incubate at room temperature for 10 minutes to stabilize luminescent signal.
8. Record luminescence.

Induce the expression of GSDMD-N275

Intracellular environment-dependent expression

P_sifA is a intracellular environment-dependent promoter. SipD is required for bacterial internalization. ΔsipD mutant cannot enter host cells. Thus, we use this strain as a control. Microscopy suggested that only intracellular bacteria express eGFP which under the control of P_sifA (Figure 8).

Figure 8. Microscopy of hela GSDMD KO cells infected with the ΔsipD or ΔsifA mutant, respectively. These mutants contain low copy number plasmids to express eGFP which was regulated by P_sifA

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 Bacterial Cells
1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture 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 °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 bacterial cells (MOI = 100) by adding bacterial cells 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.

ATc-dependent expression

GSDMD-N275 can 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 P_tet. The colony-forming unit (CFU) was measured for counting the number of viable bacterial cells (Figure 9). This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.

Figure 9. 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 bacterium grown to logarithmic phase (OD = 0.6~0.8). Vector refers to bacterium containing a high copy number plasmid which only express TetR under the control of P_tet. CFU for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3).

Method

1. Bacteria 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.

GSDMD-N275 from lytic bacteria induces host 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 (16μg/mL) were added 3h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and triggered pyroptosis after 30 min of induction (Figure 10). After 1.5 h of induction, Hela GSDMD KO cells underwent second necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis⁴. Thus, the population of ruptured cells was counted. There is 2-fold change between control group and induced group (Figure 11). So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial infection.

In these experiment, the choice of MOI and infection time were conducted by Modelling.

Figure 10. Hela GSDMD KO cells were infected with ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photographs were captured 5 min, 30 min, 90 min after induction, respectively.

Figure 11. Numbers of pyroptotic cells before and after ATc induction. 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.

Reference

1 Dumont, A. et al. SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges. Traffic 11, 899-911, doi:10.1111/j.1600-0854.2010.01069.x (2010).

2 Thurston, T. L. et al. Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death. Nature communications 7, 13292, doi:10.1038/ncomms13292 (2016).

3 Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat Protoc 10, 1820-1841, doi:10.1038/nprot.2015.115 (2015).

4 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

Chassis

Targeting

Function of GSDMD

Induce the expression of GSDMD-N275

Reference

Back to Top

Demonstrate

@@ Line 808: / Line 808: @@
          <div class="zhengwen">
              <div id="float01" class="cur">
-                <div class="h1">Chassis</div>
+                 <div class="h1">Chassis</div>
                  <div class="h2"><i>sifA</i> knock out</div>
                  <p>SifA maintains the integrity of <i>Salmonella</i>-containing vacuole (SCV) where <i>Salmonella</i>
@@ Line 825: / Line 825: @@
                      <img src="https://static.igem.org/mediawiki/2018/7/79/T--HZAU-China--results1.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 1.</b> PCR verification of <i>sifA</i> gene knock out. Lane 1 refers to mutant candidate.
+                 <p><b>Figure 1.</b> PCR verification of <i>sifA</i> gene knock out. Lane 1 refers to Δ<i>sifA</i> mutant.
                      Lane 2 refers
-                     to WT <i>Salmonella enterica</i> var. Typhimurium SL1344 as a control.</p>
+                     to WT <i>Salmonella enterica</i> serovar Typhimurium strain SL1344 as a control.</p>
                  <div style="width: 90%; margin: 20px auto">
                      <img src="https://static.igem.org/mediawiki/2018/0/07/T--HZAU-China--results2.png" width=100% alt="">
@@ Line 836: / Line 836: @@
                  <div class="h2">Safety</div>
-                 <p>SifA is essential for maintaining vacuolar membrane stability. Cytosolic bacteria can be generated
+                 <p>SifA is essential for maintaining vacuolar membrane stability. Cytosolic bacterium can be generated
-                     by the function loss of <i>Salmonella</i>-containing vacuole (SCV). This population of bacteria was
+                     by the function loss of SCV. This population of bacterium was
                      easily
                      recognized and cleaned up by macrophage. Hence, Δ<i>sifA</i> mutant decrease the toxicity to the
@@ Line 849: / Line 849: @@
                  <p><b>Figure 3.</b> Microscopy of immortalized bone-marrow-derived macrophages (iBMDM) infected with
                      the Δ<i>sifA</i>
-                     mutant and WT <i>Salmonella</i>, respectively. These strains contain high copy number plasmids to
+                     mutant and WT <i>Salmonella</i> SL1344, respectively. These strains contain high copy number plasmids to
                      express RFP constitutively.</p>
                  <div class="collapseDiv">
@@ Line 857: / Line 857: @@
                          <b>Preparation of Cells for Infection</b><br>
 . Grow iBMDMs in a humidified 37 °C, 5% CO<sub>2</sub> tissue-culture incubator.<br>
-. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow
+. Count the cells using a hemocytometer. Seed in 24-well (5×10^4 per well) and grow
                          overnight .<br>
-                         <b>Preparation of Bacteria</b>
+                         <b>Preparation of the Bacterial Cells</b>
-. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
+. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
                          incubator (200 rpm).<br>
-. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a
+. Subculture 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 °C in a shaking incubator (200 rpm) to late log
                          phase.<br>
-. Pellet 1 mL of the <i>Salmonella</i> subculture by centrifugation at 1000 g in a microfuge
+. Pellet 1 mL of the <i>Salmonella</i> bacteria cells subculture by centrifugation at 1,000×g in a microfuge
                          for 2
                          min at room temperature.<br>
@@ Line 871: / Line 871: @@
                          <b>Infection</b>
 . Aspirate media and rinse the monolayer twice with PBS.<br>
-. Inoculate cells with bacteria (MOI = 20) by adding bacteria directly to the cell-culture
+. Inoculate cells with bacterial cells (MOI = 20) by adding bacterial cells directly to the cell-culture
-                         supernatant. Centrifugation at 110 g for 5 min at room temperature.<br>
+                         supernatant. Centrifugate at 110 g for 5 min at room temperature.<br>
 . Incubate for 25 min at 37 °C in 5% CO<sub>2</sub>.<br>
-. Aspirate media and rinse the monolayer twice with PBS, to remove extracellular bacteria.<br>
+. Aspirate media and rinse the monolayer twice with PBS, to remove extracellular bacterial cells.<br>
 . Add fresh GM containing 100 μg/mL gentamicin and incubate for 2 h at 37 °C in 5% CO<sub>2</sub>.<br>
 . Replace GM with fresh GM containing 20 μg/mL gentamicin for remainder of experiment.<br>
@@ Line 886: / Line 886: @@
                  <p>RGD motif can specifically bind to alpha V beta 3 (αVβ3), a well-known biomarker on the surface of
                      tumor cells. We express RGD motif fused with OmpA to surface display it on the outer membrane of
-                     bacteria. Microscopy shows that bacteria expressed Lpp-OmpA-RGD induced by 0.1 mM IPTG can bind to
+                     bacterial cells. Microscopy shows that bacterial cells expressed Lpp-OmpA-RGD induced by 0.1 mM IPTG can bind to
-                     αVβ3-positive MDA-MB-231 cell line. The location of bacteria is pointed by red arrow (<b>Figure 4</b>).
+                     αVβ3-positive MDA-MB-231 cell line. The location of bacterial cells is pointed by red arrow (<b>Figure 4</b>).
                      But cannot bind to αVβ3-negative MCF7 cell line (<b>Figure 5</b>). These results demonstrated that
-                     bacteria gain the function of tumor targeting though display RGD motif. </p>
+                     the bacterium gain the function of tumor targeting though display RGD motif. </p>
                  <div style="width: 90%; margin: 0px auto">
                      <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width=100% alt="">
@@ Line 895: / Line 895: @@
                  <p><b>Figure 4.</b> Microscopy of αVβ3-positive MDA-MB-231 cell line were incubated with <i>E. coli</i>
                      which
-                     constructive expressed RFP and inductively expressed RGD motif under the control of lac promoter. </p>
+                     constructive expressed RFP and inductively expressed RGD motif under the control of P<sup><i>lac</i></sup>. </p>
                  <div style="width: 90%; margin: 0px auto">
                      <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width=100% alt="">
@@ Line 910: / Line 910: @@
 . Count the cells using a hemocytometer. Seed in 24-well (1.5× 10^5 per well) and grow
                          overnight.<br>
-                         <b>Preparation of Bacteria</b>
+                         <b>Preparation of Bacterial cells</b>
-. Grow bacteria overnight 14 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
+. Grow bacterial cells overnight 14 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
                          incubator (200 rpm).<br>
-. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB
+. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB
                          containing 0.1 mM IPTG in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator
                          (200 rpm) to early stationary phase.<br>
-. Pellet 1 mL of the bacteria subculture by centrifugation at 1000 g in a microfuge for 2 min
+. Pellet 1 mL of the bacterial cells subculture by centrifugation at 1000 g in a microfuge for 2 min
                          at room temperature.<br>
 . Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
                          <b>Infection</b>
 . Aspirate media and rinse the monolayer twice with PBS.<br>
-. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture
+. Inoculate cells with bacterial cells (MOI = 100) by adding bacterial cells directly to the cell-culture
                          supernatant.<br>
 . Incubate for 1.5 h at 37 °C in 5% CO<sub>2</sub>.<br>
@@ Line 932: / Line 932: @@
                  <div class="h1">Function of GSDMD</div>
                  <div class="h2">The N-terminal of GSDMD performs the function of cell pyroptosis</div>
-                 <p>We fused eGFP with GSDMD-N275 and GSDMD FL (full length), respectively. Then the corresponding
+                 <p>We fused eGFP with GSDMD-N275 (N-terminal 275 amino acid) 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 6</b>).
-                     We also tested the cell viability through an ATP assay (CellTiter-Glo® Luminescent Cell Viability
+                     We also tested the cell viability through 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
+                     Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different abilities to induce
                      pyroptosis (<b>Figure 7</b>).</p>
                  <div style="width: 90%; margin: 0px auto">
                      <img src="https://static.igem.org/mediawiki/2018/7/7b/T--HZAU-China--results6.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 6.</b> Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL and
+                 <p><b>Figure 6.</b> Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL (above) and
-                     pCS2-eGFP-GSDMD-N275, respectively. Pyroptotic cells are pointed by red arrow.</p>
+                     pCS2-eGFP-GSDMD-N275 (left), respectively. Pyroptotic cells are pointed by red arrow.</p>
                  <div class="collapseDiv">
                      <label for="zhedie-toggle3">Method</label>
@@ Line 951: / Line 951: @@
 . 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>
 . Incubate for 10 min at RT.<br>
@@ Line 980: / Line 980: @@
 . Equilibrate the plate and its contents at room temperature for approximately 30 minutes
                          after 20 h.<br>
-. Add a volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium present in
+. Add a volume of CellTiter-Glo<sup>®</sup> Reagent equal to the volume of cell culture medium present in
                          each well. (add 100 μl of reagent to 100 μl of medium containing cells for a 96-well plate).<br>
 . Mix contents for 2 minutes on an orbital shaker to induce cell lysis.<br>
@@ Line 1,012: / Line 1,012: @@
 . Count the cells using a hemocytometer. Seed in 24-well (9× 10^4 per well) and grow
                          overnight.<br>
-                         <b>Preparation of Bacteria</b> <br>
+                         <b>Preparation of Bacterial Cells</b> <br>
-. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
+. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
                          incubator (200 rpm).<br>
-. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a
+. Subculture 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 °C in a shaking incubator (200 rpm) to late log
                          phase.<br>
@@ Line 1,024: / Line 1,024: @@
                          <b>Infection</b> <br>
 . Aspirate media and rinse the monolayer twice with PBS.<br>
-. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture
+. Inoculate cells with bacterial cells (MOI = 100) by adding bacterial cells directly to the cell-culture
                          supernatant.<br>
 . Incubate for 3 h at 37 °C in 5% CO<sub>2</sub>.<br>
@@ Line 1,037: / Line 1,037: @@
                      serovar Typhimurium str. SL1344 Δ<i>sifA</i> is under the control of P<sub>tet</sub>. The
                      colony-forming unit (CFU)
-                     was measured for counting the number of viable bacteria (<b>Figure 9</b>). This result shows that
+                     was measured for counting the number of viable bacterial cells (<b>Figure 9</b>). This result shows that
                      eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.</p>
                  <div style="width: 40%; margin: 0px auto">
@@ Line 1,044: / Line 1,044: @@
                  <p><b>Figure 9.</b> CFU comparison between the SL1344 Δ<i>sifA</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
+                     empty vector. In each group, ATc (15μg/ml) was added into medium when bacterium grown to logarithmic
-                     phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only
+                     phase (OD = 0.6~0.8). Vector refers to bacterium containing a high copy number plasmid which only
-                     express TetR under the control of P<sub>tet</sub>. Bacterial colony-forming units (CFU) for vector
+                     express TetR under the control of P<sub>tet</sub>. CFU for vector
                      and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3).</p>
                  <div class="collapseDiv">
@@ Line 1,067: / Line 1,067: @@
                      promoter in Δ<i>sifA</i> SL1344. Hela GSDMD KO cells 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
+                     (16μg/mL) were added 3h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm
-                     after 5 min of induction and trigger pyroptosis after 30 min of induction (<b>Figure 10</b>). After
+                     after 5 min of induction and triggered pyroptosis after 30 min of induction (<b>Figure 10</b>). After
 .5 h
-                     of induction, Hela GSDMD KO cells undergo second necrosis caused by bacterial infection without
+                     of induction, Hela GSDMD KO cells underwent second necrosis caused by bacterial infection without
                      inducer. Morphology of this process is similar to pyroptosis<sup>4</sup>. Thus, the population of
                      ruptured
@@ Line 1,083: / Line 1,083: @@
                  <p><b>Figure 10.</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
+                     which express eGFP-GSDMD-N275 under the control of ATc. Photographs were captured 5 min, 30 min, 90 min
                      after induction, respectively.</p>
                  <div style="width: 40%; margin: 0px auto">