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

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                     transfected into Hela GSDMD KO cell. Microscopy of cells transfecting GSDMD-N275 undergoing
 
                     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
 
                     pyroptosis, but GSDMD full length did not induce pyroptosis (Figure 1). We also tested the cell
−
                     viability through ATP assay (CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay) and demonstrated that
+
                     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>
 
                     GSDMD-N275 and mutants of GSDMD FL had different ability to induce pyroptosis (Figure 2).</p>
 
                 <div style="width: 40%; margin: 30px auto">
 
                 <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>
 
                 </div>
−
                 <p>Figure 1. pCS2-eGFP-GSDMD FL (left), pCS2-eGFP-GSDMD-N275 (right) were transfected respectively into
+
                 <p>Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into
−
                     Hela GSDMD KO cells. Pyroptotic cells are pointed by red arrow.</p>
+
                     Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.</p>
 
                 <div class="collapseDiv">
 
                 <div class="collapseDiv">
 
                     <label for="zhedie-toggle1">Method</label>
 
                     <label for="zhedie-toggle1">Method</label>
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                     <div id="zhedie1" class="text-success text-left">
 
                     <div id="zhedie1" class="text-success text-left">
 
                         <b>Preparation of Cells for transfection</b><br>
 
                         <b>Preparation of Cells for transfection</b><br>
−
                         1. Grow Hela GSDMD KO cells in a humidified 37℃, 5% CO<sub>2</sub> tissue-culture
+
                         1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO<sub>2</sub> tissue-culture
 
                         incubator.<br>
 
                         incubator.<br>
−
                         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>
−
                         1. Dilute 0.5 μg DNA into 50 μl jetPRIME<sup>®</sup> buffer (supplied). Mix by vortexing.<br>
+
                         1. Dilute 0.5 μg DNA into 50 μl jetPRIME® buffer (supplied). Mix by vortexing.<br>
−
                         2. Add 1 μl jetPRIME<sup>®</sup>, vortex for 10 s, spin down briefly.<br>
+
                         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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                     <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>
 
                 </div>
−
                 <p>Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373D,
+
                 <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
 
                     pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was
 
                     measured (n=6).</p>
 
                     measured (n=6).</p>
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                     <div id="zhedie2" class="text-success text-left">
 
                     <div id="zhedie2" class="text-success text-left">
 
                         <b>Preparation of Cells for Infection</b><br>
 
                         <b>Preparation of Cells for Infection</b><br>
−
                         1. Grow Hela GSDMD KO cells in a humidified 37℃, 5% CO<sub>2</sub> tissue-culture
+
                         1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO<sub>2</sub> tissue-culture
 
                         incubator.<br>
 
                         incubator.<br>
 
                         2. Count the cells using a hemocytometer. Seed in 24-well (9× 10^4 per well) and grow
 
                         2. Count the cells using a hemocytometer. Seed in 24-well (9× 10^4 per well) and grow
 
                         overnight.<br>
 
                         overnight.<br>
 
                         <b>Preparation of Bacteria</b><br>
 
                         <b>Preparation of Bacteria</b><br>
−
                         1. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37℃ in a shaking
+
                         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).<br>
 
                         incubator (200 rpm).<br>
 
                         2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a
 
                         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℃ in a shaking incubator (200 rpm) to late log
+
                         loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log
 
                         phase.<br>
 
                         phase.<br>
−
                         3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1,000 ×g in a microfuge for
+
                         3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 g in a microfuge for
 
                         2 min at room temperature.<br>
 
                         2 min at room temperature.<br>
 
                         4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
 
                         4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
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                         2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the
 
                         2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the
 
                         cell-culture supernatant.<br>
 
                         cell-culture supernatant.<br>
−
                         3. Incubate for 3 h at 37℃ in 5% CO<sub>2</sub>.<br>
+
                         3. Incubate for 3 h at 37 °C in 5% CO<sub>2</sub>.<br>
 
                         4. Aspirate media and rinse the monolayer twice with PBS.<br>
 
                         4. Aspirate media and rinse the monolayer twice with PBS.<br>
−
                         5. Add fresh GM containing 100 μg/mL gentamicin and incubate at 37℃ in 5% CO<sub>2</sub>.<br>
+
                         5. Add fresh GM containing 100 μg/mL gentamicin and incubate at 37 °C in 5% CO<sub>2</sub>.<br>
 
                         Observation is taken after 2 h.<br>
 
                         Observation is taken after 2 h.<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>
 
                 <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) was under the control of P<sub>tet</sub>
−
                     in <i>ΔsifA</i> SL1344.
+
                     in Δ<i>sifA</i> SL1344.
 
                     The colony-forming unit (CFU) was measured for counting the number of viable bacteria (Figure 3).
 
                     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>
 
                     This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.</p>

Revision as of 11:35, 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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