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

Revision as of 11:03, 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.

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. 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).

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) was under the control of P_tet 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 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) 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.

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

Back to Top

Composite Part

@@ Line 346: / Line 346: @@
              line-height: 80px;
              font-weight: bold;
+            height:auto;
              font-family: 'Product Sans', sans-serif;
              font-size: 40px;
@@ Line 357: / Line 358: @@
              line-height: 40px;
              font-weight: bold;
+            height:auto;
              /* font-weight: bold; */
              font-family: 'Product Sans', sans-serif;
@@ Line 368: / Line 370: @@
              line-height: 30px;
              font-weight: bold;
+            height:auto;
              /* font-weight: bold; */
              font-family: 'Product Sans', sans-serif;
@@ Line 787: / Line 790: @@
                      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<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>
                  <div style="width: 40%; margin: 30px auto">