iGEM 2018 — Team HZAU-China

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# Team:HZAU-China/Notebook

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  [Human Practices](https://2018.igem.org/Team%3AHZAU-China/Human_Practices)
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Processing of experiment results

## **1.** Verification of Poisson distribution

We infect tumor cells (HeLa GSDMD KO cell line) with *Salmonella* carrying the GFP gene for a period of time and
then count them, and the result is that the distribution of *Salmonella* in the tumor cells
is consistent with a Poisson distribution.

![](https://static.igem.org/mediawiki/2018/8/86/T--HZAU-China--Notebook-fig1.png)

**Figure 1.** Infection of HeLa GSDMD KO cells by *Salmonella* and the result of analysis.

The amounts of host cells and bacteria were measured in experiments.

The concentration of bacteria was calculated according to MOI (multiplicity of infection) and cell density.

We adjusted the parameter As to match the curve with the results of the Salmonella infection experiment. When As is
equal to 0.0003, the curve simulated by the mathematical model matches the experimental measurements.

![](https://static.igem.org/mediawiki/2018/8/84/T--HZAU-China--Notebook-fig2.png)

**Figure 2.** Parameter fitting of the tumor
cells infection experiments. Red points are experimental results.

The parameters ‘Nsal’ and ‘Tumor’ are already known before the experiment. When the parameter ‘As’ is 0.0003,
the model results best fit the experimental results.

[Experiments
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# Team:HZAU-China/Experiments

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Material

**1.** Bacteria Strains used in this work

| Strain | Description | Usage | Sourse |
| --- | --- | --- | --- |
| *E.coli* trans-5α | Basic strain of calcium conversion | Plasmid construction, molecular cloning | Purchase by Shanghai Weidi Biotechnology |
| *E.coli* MG1655 | Type strain | Phenotypic validation | Provide by Dr. Chenli Liu (SIAT CSynBER) |
| *S.* typhimurium SL1344 | Type strain | Phenotypic validation | Provide by Dr. Shan Li (Bio-Medical Center of HZAU) |
| *S.* typhimurium SL1344 (*ΔsifA*） | Decrease toxicity and infectivity | Phenotypic validation | This work |
| *S.* typhimurium SL1344 (*ΔsipD*） | Knockout Type III secretion system | Phenotypic validation | This work |

**2.** Culture Condition

| Culture medium | Compositions |
| --- | --- |
| Luria Broth (LB) | 0.5% yeast extraction,1% NaCl and 1% tryptone (add 15 g/L agar when prepare solid culture) |
| Super Optimal Broth (SOB) | 0.5% yeast extraction, 0.05% NaCl and 2% tryptone (add 15 g/L agar when prepare solid culture) (add 5ml 2 mol/L MgCl2 before use) |

Method

**1.** Plasmid construction

Our fragments was PCR amplified with KOD (TOYOBO®) or PrimeSTAR (Takara®)
according to product length.

Product is recycled with gel extraction kit from OMEGA® after electrophoresis.

ClonExpress® II One Step Cloning Kit (Vyzyme) was used to ligate every fragment to
construct the most plasmids.

All plasmid constructs were conﬁrmed by sequencing at Sangon®, Inc. (Wuhan,China).

A single frozen glycerol stock was used throughout this study for each bacterial strain.

**2.** Transformation

1. Add 3-5μl plasmid to 50μl F-trans5α (Weidi Biotechnology) competent cells and incubate 5min in ice
, coated plates, select monoclonal colony PCR. Add extra 42℃ heat shock 45s and incubate 10min step
will increase transformation efficiency.
Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950 rpm for 1h.

2. Plated on LB agar plates containing appropriate concentration of antibody for selection, grown
overnight at 37°C. And prepare PCR reaction to select individual bacterial colony.

**3.** Fluorescence/growth measurememt

Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted to OD is
0.1 with fresh LB broth.

Expression was induced at early log phase by addition of different ATc (150 ng/ml–15μg/ml)
concentrations. Culture the plate in 37℃, 150 rpm. Every hour put it into a plate reader to
measure its fluorescence/OD1.

Modeling impact our experiment

The higher ATc level might thwart bacterial growth. So we need to analyze a proper concentration of
ATc, We design a modeling to predict a suitable concentration of ATc (More result about our model
please check [modeling: Chemical control model](https://2018.igem.org/Team%3AHZAU-China/Model)).
We induced our engineered strain with gradients of ATc. Take 100μl culture to plate on LB agar
plates containing appropriate concentration of antibody after 5 minutes, 30 minutes, 90 minutes of
induce and counting Colony Forming Units the next day. The results fit the model within a range of
ATc. So we choose a appropriate concentration of ATc (16μg/ml), aiming to maximize the expression of
GSDMD-N275 and minimize the negative effect of bacteria growth.

Reference

1. Becskel, A. & Serrano, L. Engineering stability in gene networks by autoregulation. Nature 405,
590–593 (2000).

Experiments
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Material

Method

Modeling impact our experiment

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# Team:HZAU-China/Results

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  [Composite](https://2018.igem.org/Team%3AHZAU-China/Composite_Part)

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 Δ*sifA* mutant in macrophage is remarkable2. Thus, we
knocked out the
*sifA* gene in order to decrease the stability of SCV and reduce the virulence of *Salmonella*.

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

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

![](https://static.igem.org/mediawiki/2018/7/79/T--HZAU-China--results1.png)

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

![](https://static.igem.org/mediawiki/2018/0/07/T--HZAU-China--results2.png)

**Figure 2.** DSIFA F and DSIFA R are the primers in the ORF of *sifA*. This pair of primers
can produce 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, Δ*sifA* mutant decreases the toxicity to the
host.
Microscopy demonstrated that Δ*sifA* mutant was defective for replication in macrophage (**Figure
3**).

![](https://static.igem.org/mediawiki/2018/9/93/T--HZAU-China--results3.png)

**Figure 3.** Microscopy of immortalized bone-marrow-derived macrophages (iBMDM) infected with
the Δ*sifA*
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 (**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.

![](https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png)

**Figure 4.** Microscopy of αvβ3-positive MDA-MB-231 cell line were incubated with *E. coli*
which
constructively expressed RFP and inductively expressed RGD motif under the control of *lac* promoter. The locations of bacteria cells are pointed by red arrow.

![](https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png)

**Figure 5.** Microscopy of αvβ3-negative MCF7 cell line were incubated with *E. coli* which
constructively
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 acids) and GSDMD FL (full length), respectively. Then the corresponding
plasmids were transfected into Hela GSDMD knockout (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**).

![](https://static.igem.org/mediawiki/2018/d/d7/T--HZAU-China--basicPart1.png.png)

**Figure 6.** Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL (above) and
pCS2-eGFP-GSDMD-N275 (below), 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.

![](https://static.igem.org/mediawiki/2018/f/f5/T--HZAU-China--basicPart2.png)

**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

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

![](https://static.igem.org/mediawiki/2018/0/0b/T--HZAU-China--results8.png)

**Figure 8.** Microscopy of hela GSDMD KO cells infected with the Δ*sipD* or Δ*sifA*
mutant, respectively.
These mutants contain low copy number plasmids to express eGFP which was regulated by PsifA.

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 lyse bacteria

Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) in *Salmonella enterica*
serovar Typhimurium str. SL1344 Δ*sifA* is under the control of Ptet. 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.

![](https://static.igem.org/mediawiki/2018/f/fb/T--HZAU-China--basicPart3.jpg)

**Figure 9.** CFU comparison between the SL1344 Δ*sifA* cells with eGFP-GSDMD-N275 plasmid
and with the
empty vector. In each group, ATc (15μg/ml) was added into medium when bacterium grew to logarithmic
phase (OD = 0.6~0.8). Vector refers to bacterium containing a high copy number plasmid which only
expresses TetR under the control of Ptet. 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 Δ*sifA* SL1344. Hela GSDMD KO cells were infected with Δ*sifA* 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 1.96 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 experiments, the choice of MOI (multiplicity of infection) and infection time were directed by [Modeling](https://2018.igem.org/Team%3AHZAU-China/Model).

![](https://static.igem.org/mediawiki/2018/b/b3/T--HZAU-China--basicPart4.png)

**Figure 10.** Hela GSDMD KO cells were infected with Δ*sifA* 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.

![](https://static.igem.org/mediawiki/2018/2/22/T--HZAU-China--basicPart5.png)

**Figure 11.** 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 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 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, 90min.

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,
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[Description
![](https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg)](https://2018.igem.org/Team%3AHZAU-China/Description)
[Design
![](https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg)](https://2018.igem.org/Team%3AHZAU-China/Design)

Results
![](https://static.igem.org/mediawiki/2018/8/8d/T--HZAU-China--xjt.svg)

Chassis

Targeting

Function of GSDMD

Induce the expression of GSDMD-N275

Reference

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[Demonstrate
![](https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg)](https://2018.igem.org/Team%3AHZAU-China/Demonstrate)