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

Revision as of 14:48, 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 mutant candidate. Lane 2 refers to WT Salmonella enterica var. Typhimurium 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 bacteria can be generated by the function loss of Salmonella-containing vacuole (SCV). This population of bacteria 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, 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 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 = 20) by adding bacteria directly to the cell-culture supernatant. Centrifugation 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 bacteria.
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 bacteria. Microscopy shows that bacteria 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 (Figure 4). But cannot bind to αVβ3-negative MCF7 cell line (Figure 5). These results demonstrated that bacteria 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 inductive expressed RGD motif under the control of lac promoter.

Figure 5. Microscopy of αVβ3-negative MCF7 cell line were incubated with E. coli which constructive expressed RFP and inductive 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 Bacteria 1. Grow bacteria overnight 14 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 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 bacteria 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 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 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 ability to induce pyroptosis (Figure 7).

Figure 6. 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 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 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.

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 bacteria (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 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. 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 locates in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Figure 10). 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 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. Photos were captured 5 min, 30min, 1.5h 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 810: / Line 810: @@
                  <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> survive and
+                 <p>SifA maintains the integrity of <i>Salmonella</i>-containing vacuole (SCV) where <i>Salmonella</i>
-                     replicate<sup>1</sup>. The existence of SCV limits the releasing of GSDMD-N275 into cytoplasm. In addition,
+                    survive and
-                     the growth of inhibition of Δ<i>sifA</i> mutant in macrophage is remarkable<sup>2</sup>. Thus, we knocked out the
+                     replicate<sup>1</sup>. The existence of SCV limits the releasing of GSDMD-N275 into cytoplasm. In
-                     <i>sifA</i> gene in order to prevent the stability of SCV and reduce the virulence of <i>Salmonella</i>. </p>
+                    addition,
+                     the growth of inhibition of Δ<i>sifA</i> mutant in macrophage is remarkable<sup>2</sup>. Thus, we
+                    knocked out the
+                     <i>sifA</i> gene in order to prevent the stability of SCV and reduce the virulence of <i>Salmonella</i>.
+                </p>
                  <p>Δ<i>sifA</i> mutant was constructed by using gene editing system based on two-step allelic exchange<sup>3</sup>.</p>
-                 <p>PCR verification indicated that chromosomal gene <i>sifA</i> was knocked out (<b>Figure 1</b>). Primers named
+                 <p>PCR verification indicated that chromosomal gene <i>sifA</i> was knocked out (<b>Figure 1</b>).
+                    Primers named
                      DSIFA F and DSIFA R were used in this PCR (<b>Figure 2</b>).</p>
                  <div style="width: 40%; margin: 20px auto">
                      <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. Lane 2 refers
+                 <p><b>Figure 1.</b> PCR verification of <i>sifA</i> gene knock out. Lane 1 refers to mutant candidate.
+                    Lane 2 refers
                      to WT <i>Salmonella enterica</i> var. Typhimurium 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="">
                  </div>
-                 <p><b>Figure 2.</b> DSIFA F and DSIFA R are the primers in the ORF of <i>sifA</i>. This pair of primers can extend a
+                 <p><b>Figure 2.</b> DSIFA F and DSIFA R are the primers in the ORF of <i>sifA</i>. This pair of primers
+                    can extend a
 bp product in WT.</p>
                  <div class="h2">Safety</div>
                  <p>SifA is essential for maintaining vacuolar membrane stability. Cytosolic bacteria can be generated
-                     by the function loss of <i>Salmonella</i>-containing vacuole (SCV). This population of bacteria was easily
+                     by the function loss of <i>Salmonella</i>-containing vacuole (SCV). This population of bacteria was
-                     recognized and cleaned up by macrophage. Hence, Δ<i>sifA</i> mutant decrease the toxicity to the host.
+                    easily
-                     Microscopy demonstrated that Δ<i>sifA</i> mutant was defective for replication in macrophage (<b>Figure 3</b>).
+                     recognized and cleaned up by macrophage. Hence, Δ<i>sifA</i> mutant decrease the toxicity to the
+                    host.
+                     Microscopy demonstrated that Δ<i>sifA</i> mutant was defective for replication in macrophage (<b>Figure
+</b>).
                  </p>
                  <div style="width: 90%; margin: 0px auto">
                      <img src="https://static.igem.org/mediawiki/2018/9/93/T--HZAU-China--results3.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 3.</b> Microscopy of immortalized bone-marrow-derived macrophages (iBMDM) infected with the Δ<i>sifA</i>
+                 <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
                      express RFP constitutively.</p>
@@ Line 854: / Line 865: @@
                          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 for 2
+. Pellet 1 mL of the <i>Salmonella</i> 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>
@@ Line 881: / Line 893: @@
                      <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 4.</b> Microscopy of αVβ3-positive MDA-MB-231 cell line were incubated with <i>E. coli</i> which
+                 <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 inductive expressed RGD motif under the control of lac promoter. </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="">
                  </div>
-                 <p><b>Figure 5.</b> Microscopy of αVβ3-negative MCF7 cell line were incubated with <i>E. coli</i>  which constructive
+                 <p><b>Figure 5.</b> Microscopy of αVβ3-negative MCF7 cell line were incubated with <i>E. coli</i> which
+                    constructive
                      expressed RFP and inductive expressed RGD motif under the control of lac promoter.</p>
                  <div class="collapseDiv">
@@ Line 980: / Line 994: @@
                  <p>P<sub>sifA</sub> is a intracellular environment-dependent promoter. SipD is required for bacterial
                      internalization.
-                     Δ<i>sipD</i> mutant cannot enter host cells. Thus, we use this strain as a control. Microscopy suggested
+                     Δ<i>sipD</i> mutant cannot enter host cells. Thus, we use this strain as a control. Microscopy
-                     that only intracellular bacteria express eGFP which under the control of P<sub>sifA</sub> (<b>Figure 8</b>). </p>
+                    suggested
+                     that only intracellular bacteria express eGFP which under the control of P<sub>sifA</sub> (<b>Figure
+</b>). </p>
                  <div style="width: 90%; margin: 0px auto">
                      <img src="https://static.igem.org/mediawiki/2018/0/0b/T--HZAU-China--results8.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 8.</b> Microscopy of hela GSDMD KO cells infected with the Δ<i>sipD</i> or Δ<i>sifA</i> mutant, respectively.
+                 <p><b>Figure 8.</b> Microscopy of hela GSDMD KO cells infected with the Δ<i>sipD</i> or Δ<i>sifA</i>
+                    mutant, respectively.
                      These mutants contain low copy number plasmids to express eGFP which was regulated by P<sub>sifA</sub></p>
                  <div class="collapseDiv">
@@ Line 1,001: / Line 1,018: @@
                          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 for 2
+. Pellet 1 mL of the <i>Salmonella</i> 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>
@@ Line 1,017: / Line 1,035: @@
                  <div class="h3">GSDMD-N275 can lyses bacteria</div>
                  <p>Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) in <i>Salmonella enterica</i>
-                     serovar Typhimurium str. SL1344 Δ<i>sifA</i> is under the control of P<sub>tet</sub>. The colony-forming unit (CFU)
+                     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
                      eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.</p>
@@ Line 1,023: / Line 1,042: @@
                      <img src="https://static.igem.org/mediawiki/2018/f/fb/T--HZAU-China--basicPart3.jpg" width=100% alt="">
                  </div>
-                 <p><b>Figure 9.</b> CFU comparison between the SL1344 Δ<i>sifA</i> cells with eGFP-GSDMD-N275 plasmid and with the
+                 <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
                      phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only
@@ Line 1,045: / Line 1,065: @@
                  <div class="h3">GSDMD-N275 from lytic bacteria induces host cell pyroptosis</div>
                  <p>Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet
-                     promoter in Δ<i>sifA</i> SL1344. Hela GSDMD KO cells were infected with Δ<i>sifA</i> SL1344. Inducer ATc
+                     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
-                     after 5 min of induction and trigger pyroptosis after 30 min of induction (<b>Figure 10</b>). After 1.5 h
+                     after 5 min of induction and trigger 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
-                     inducer. Morphology of this process is similar to pyroptosis<sup>4</sup>. Thus, the population of ruptured
+                     inducer. Morphology of this process is similar to pyroptosis<sup>4</sup>. Thus, the population of
-                     cells was counted. There is 2-fold change between control group and induced group (<b>Figure 11</b>). So
+                    ruptured
+                     cells was counted. There is 2-fold change between control group and induced group (<b>Figure 11</b>).
+                    So
                      the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial
                      infection.</p>
-<p>In these experiment, the  choice of MOI and infection time were conducted by <a href="https://2018.igem.org/Team:HZAU-China/Model">Modelling</a>.</p>
+                <p>In these experiment, the choice of MOI and infection time were conducted by <a href="https://2018.igem.org/Team:HZAU-China/Model">Modelling</a>.</p>
                  <div style="width: 90%; margin: 0px auto">
                      <img src="https://static.igem.org/mediawiki/2018/b/b3/T--HZAU-China--basicPart4.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 10.</b> Hela GSDMD KO cells were infected with Δ<i>sifA</i> SL1344 containing high copy number plasmids
+                 <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
                      after induction, respectively.</p>
@@ Line 1,063: / Line 1,088: @@
                      <img src="https://static.igem.org/mediawiki/2018/2/22/T--HZAU-China--basicPart5.png" width=100% alt="">
                  </div>
-                 <p><b>Figure 11.</b> Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of
+                 <p><b>Figure 11.</b> Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a
+                    field of
                      view were counted. </p>
                  <div class="collapseDiv">
@@ Line 1,079: / Line 1,105: @@
                          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 for 2
+. Pellet 1 mL of the <i>Salmonella</i> 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>
@@ Line 1,099: / Line 1,126: @@
                      doi:10.1111/j.1600-0854.2010.01069.x (2010).
                  </p>
-                 <p>2 Thurston, T. L. et al. Growth inhibition of cytosolic <i>Salmonella</i> by caspase-1 and caspase-11
+                 <p>2 Thurston, T. L. et al. Growth inhibition of cytosolic <i>Salmonella</i> by caspase-1 and
+                    caspase-11
                      precedes host cell death. Nature communications 7, 13292, doi:10.1038/ncomms13292 (2016).</p>
                  <p>3 Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic
@@ Line 1,178: / Line 1,206: @@
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+                 f4 = $('#float04').offset().top;
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