iGEM 2023 — Team tsbc-sz

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**Date: 7th
August,****2023**

We
designed team nick-name,
logo, and team uniform together. In the afternoon, we also participated a laboratory safety
training session.

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Overview

Ferroptosis is a natural way for cells to
die.
There are two
independent
molecular mechanisms for iron death, namely glutathione peroxidase 4 (GPX4) and ferroptosis suppressor
protein 1 (FSP1)1,2. One of these ways is using glutathione.
Therefore, we would inhibit glutathione to make ferroptosis happen3,4. To be more specific, ferroptosis needs lipid
peroxidation to intrigue cells to die. Generally, cells would consume glutathione to convert cytotoxic
lipid peroxides to non-toxic lipid alcohols
so as to
inhibit ferroptosis.
In our project, we aimed at the Gox1 gene. TNBC cells have a high metabolic rate, so they need
sufficient nutrition such as glucose.

As shown in the Figure 1, what we do is to use the Gox1 encodes glucose oxidase to inhibits
GPX4/GSH
and FSP1/CoQ10H2, the two iron death inhibition pathways, and
interferes with lipid metabolism or REDOX balance, which can lead to iron death in various cancers and
inhibit tumor cell proliferation.

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Amplify the gene of Gox1

1)PCR

|  |  |
| --- | --- |
| Material | Volume(μL) |
| 2× Mix | 20 |
| Primer F (10 μM) | 1 |
| Primer R (10 μM) | 1 |
| DNA template | 1 |
| ddH2O | 17 |
| Total | 40 |

 PCR instrument program
settings：

|  |  |  |
| --- | --- | --- |
| Step | Temperature (℃) | Time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 sec |
| 3 | 55 | 30 sec |
| 4 | 72 | 1 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS:The step2 and step4
cycle 30 times

2)Gel
electrophoresis

a.Materiel

a)Electrophoresis chamber

b)Gel
caster

c)Comb

d)Power supply
and electrodes

e)Pipettes

f)Gel

|  |  |
| --- | --- |
| Material | Mass/volume |
| Agarose | 0.5 g |
| 1xTAE | 50 mL |
| GelRed | 5 μL |

 PS: TAE components: Tris base, acetic acid,and
EDTA

g)Gox1 DNA samples

h)Trans15K DNA marker

i)6X
Gel loading dye,
purple

b.Procedure

a)Weigh 0.5-gram agarose

b) Add it into the
prepared 50mL TAE buffer in a conical flask

c) Heat it in the
oven with medium or high heat

d) Shake it for a
while after the first heating

e) Heat it
again

f) Wait until it
cools down. Place a comb and a base in the electrophoresis device and pour the solution into
it.

g) Wait until it
turns solid.

Plasmid Extraction

|  |  |
| --- | --- |
| Material | Volume(μL) |
| *E**.coli* | 3 mL |
| SP1 buffer | 250 μL |
| SP2 buffer | 250 μL |
| SP3 buffer | 250 μL |
| Wash buffer | 1000 μL |
| Elution buffer | 40 μL |

Procedure

Extract plasmids from
pET-28a bacterial fluid

1) Add 1mL *E**.coli* into bacterial fluid

2) Centrifugate with
12000xg for 5 minutes to collect bacteria

3) After
centrifugation, pour the waste fluid

4) Pour 2mL *E**.coli* into the bacterial fluid again

5) Centrifugate
again(same as previous)

6)Add 250μL SP1 buffer, then use a pipette to blow the
fluids until the solids dissolve into the fluids

7) Add
250μL SP2 buffer, reverse the tube upside and
downside for about ten times (to release protein and degrade RNA)

8) Leave it and wait
for 2 minutes

9) Add 350μL SP3 buffer(to precipitate protein), then reverse it
again (same as previous)

10) If we could see
the protein on the surface of the liquids, centrifugate it with 12000xg for 10 minutes.

11) After
centrifugation, take the supernatant fluid and centrifugate it with 8000xg for 30 seconds

12) Add
500μL wash buffer and centrifugate it with
9000xg for 30 seconds

13) Add
500μL wash buffer again and centrifugate for 1 minute
with 9000 xg(to centrifugate all wash buffer)

14) Then add
40μL elution buffer, leave it and wait for 2
minutes

15) Centrifugate it
with 9000xg for 1 minute and test the concentration of the
plasmid.

Restriction enzyme digestion and Ligation

Restriction enzyme digestion

|  |  |
| --- | --- |
| Material | Volume(μL) |
| plasmid (pET28a) | 30 |
| NheI | 1 |
| XhoI | 1 |
| Buffer (10x) (Cutsmart) | 5 |
| ddH2O | 13 |
| Total | 50 |

|  |  |
| --- | --- |
| Material | Volume(μL) |
| DNA (Gox1) | 25 |
| NheI | 1 |
| XhoI | 1 |
| Buffer (10x) (Cutsmart) | 5 |
| ddH2O | 18 |
| Total | 50 |

Placed
them
in
a 37 ℃
metal
bath for 1 hour.

Ligation

|  |  |
| --- | --- |
| Material | Volume(μL) |
| pET28a Plasmid (digested) | 10 |
| GOX1 (digested) | 7 |
| T4 ligase | 1 |
| T4 ligase buffer | 2 |
| Total | 20 |

Procedure:

1) Add 10μL pET28a Plasmid, 7μL target gene, 1μL T4 ligase and 2μL T4 ligase buffer into a new tube

2) Put the tube into the laboratory
16℃
metal
bath for 30 minutes

3) Put it into the fridge

Heat shock transformation (DH5α/BL21)

1) Mix
50μL Competent
Cells(DH5α/BL21)
with
5μL
plasmid

2) Put it on the ice
and wait for 30 minutes

3)Heat
Shock: Transfer the mixture to a water bath or heat block set at 42°C
for 90 seconds.

4) Put it on the ice
again for 3 minutes

5) Add
900μL LB, and shake it with a shaker for 1
hour at
220rpm, 37°C

6) Centrifuge
9000rpm for 1 minute.

7) Spread it on the K+ flat and
keep at 37°C overnight.

Preparing the LB solution

|  |  |
| --- | --- |
| Type | Volume/Mass |
| Tryptone | 5 g (10 g/L) |
| Yeast Extract | 2.5 g (5 g/L) |
| NaCl | 5 g (10 g/L) |
| ddH2O | 500 mL |
| Agar(solid LB only) | 7.5 g (1.5%) |
| Kanamycin | Working concentration 50 μg/mL |
| Total volume: 500 mL(+50 μL) | |

Protein Induction

1) Put 1mL pET28a-GOX1
(BL21)
strain into 400mL LB liquid medium, then add 45μL kanamycin.

2) Add 400μL IPTG solution (1M).

3) Put it into the shaker for 16h, 16
degrees.

Resuscitation of 4T1 cells

1） The 4T1 cells are brought from Beyotime.

2） Put the cells into a water bath (37 degrees) as soon as it
is received.

3） Transfer the cell suspension to a 1.5 mL tube, then put them
into a centrifuge. (500g, 5min)

4） Aspirate the supernatant, then resuspend it in a fresh
complete culture medium and transfer it to culture vessels.

5） Put it in a CO2 incubator.

HisA and HisB Preparation

His A

|  |  |
| --- | --- |
| Material | Mass |
| Na2HPO4 (40 mM) | 1.42 g |
| NaCl (500 mM) | 14.6 g |
| Imidazole (20 mM) | 0.68 g |
| HCl (6 M) | Adjust the pH to 7.4 |
| Add water to a constant volume of 0.5 L | |

His B

|  |  |
| --- | --- |
| Material | Mass |
| Na2HPO4 (20 mM) | 0.71 g |
| NaCl (500 mM) | 7.3 g |
| Imidazole (500 mM) | 8.5 g |
| HCl (6 M) | Adjust the pH to 7.4 |
| Add water to a constant volume of 0.25 L | |

Purification

1)Protein
purification

a.

a) Take
IPTG(1mM)-induced BL21(with pET28a-Gox1) bacterial solution and divide them into 6
centrifugal tubes

b)Centrifugate it with
5000rpm for 10 minutes to collect thallus, then
discard the supernatant fluid

c) Pour
40mL bacterial fluid and repeat step (b)
again

d) Pour
10mL bacterial fluid and repeat step (b)
again

e) Add 15mL His-A buffer into the tubes

f) Use a pipette to
blow the fluid for about 15 minutes. It might need to be put in the ice for several seconds after a period
of blowing

g) Put the tube into
the insulation bucket with ice, and make sure it is fixedly placed

h)Use
ultrasonic cell crusher to crush it. (500W power, work 3s & stop 3s, 15
minutes)

b.

Use centrifugation to
collect supernatant fluid

a)Collect the fluid after
ultrasonic crash and centrifugate it at 12000 rpm for 20 minutes, under the temperature of
4℃.

b)Repeat the
centrifugation

c) Collect the
supernatant fluid, and the fluid is enzyme-containing crude suspension

2) Nickel column affinity purification

a) Collect the
bacterial fluid into collection tubes and keep a little amount as a sample(for contrast)

b) Balance the nickel
column with His-A buffer twice

c) Add the protein
solution into the nickel column, and wait until it flows to the bottom

d) Add 10mL His-B buffer to elute protein(Keep it at
4℃)

e) Use 5mL of 20% ethanol to wash the column

f) Transfer the fluid
to ultrafiltration centrifugal tubes

g) Centrifugate the
tubes and test the concentration with the micro detector

Glucose oxidase activity test

|  |  |
| --- | --- |
| Material | Volume |
| Lysate solution | 15 mL |
| Working solution | 174 μL |
| Solution C | 6 μL |
| Loading | 20 μL |

Procedure:

1)Add
15mL bacteria
solution and 15mL lysate
solution

2)Use
the extract from the glucose oxidase activity test kit to resuspend

3)Put
it on ice (ice needs to be compacted), then
use ultrasonic cell crusher to crush it at 500W power, work 3s & stop 3s, 15 minutes

4)Make
the working solution(15mLA
+ 3mLB),
then add 174μL of
working solution and 6μL solution
c.

5)Gel
electrophoresis

6)Add
20μL loading. Add
5,10,20,40,60,80 μL samples
to be tested respectively

7)80μL does
not have purified cracked protein and washed protein with His buffer.

8)Place
at 37 ℃ and
measure at 5 minutes and 2 hours

9)Microporous
template inspection system (450 nm, detection
of light absorption value)

Detect the weight of the target protein

1)The
protein solution is centrifuged in a concentration tube (12000 rpm
for 5min).The machine should
be cooled to 4°C before centrifugation.

2)
Add 20μL protein
loading to 80μL protein
solution. Put
it into boiling water for 10min.

3)Take
10μL of
the solution, and
used the micro detector to measure its concentration.

4)Gel
electrophoresis (90V for
30 min, then
120V for
30min)

5)Dye
with BBI Coomassie brilliant blue, then
place in the microwave on medium heat for three minutes

6)Coomassie
brilliant blue eluent

|  |  |
| --- | --- |
| Material | Volume |
| Acetic acid | 100 mL |
| Ethyl alcohol | 400 mL |
| ddH2O | 500 mL |

CCK-8 Test (Cell Counting Kit-8 Kit from Beyotime)

1） Put cells in a 96-well plate at a density of
100,000/cm².

2） Data: RSL/1μM: pET28a-Gox1/ 16 32 64 μg/mL

3） Incubate the solution for 24 hours, then add
10μL CCK-8

4） Place the cells in a cell culture incubator at 37 degrees
for 1 hour.

5） Measure the absorbance at 450nm.

Reduced Glutathione (GSH) Content Assay Kit

1) Put cells in a 6-well plate
at a density of 100,000/cm².

2) Data: RSL/1μM: pET28a-Gox1/ 16 32 64 μg/mL

3) Incubate the solution for 24
hours.

4) Flush with PBS once.

5) Add 1mL of reagent
1, then resuspend the cells.

6) Freeze-thaw with liquid nitrogen 2-3
times, then put it into a centrifuge. (12000 rpm, 10min)

7) Preheat the reagent 2 to 37 degrees.
(30 min)

8) Prepare 1 mg/mL standard solution
into 300µg/mL, 200µg/mL, 100µg/mL, 50µg/mL and 25µg/mL standard solution.

9) Add 20μL of standard, 140μL of reagent 2 and 40μL of reagent 3.

10) Homogenize the reagent, then measure
the absorbance at 405nm.

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Overview

We aimed at
introducing the Gox1 protein for TNBC treatment which will digest glucose in order to cut the nutrition
income of TNBC so as to make ferroptosis happen. In our project, we constructed the plasmid
pET-28a-Gox1and pCOLA-CI857-SRRz-GOX1, obtained the protein upon inducing expression. Following that we
used glucose oxidase activity detection assay, reduced glutathione (GSH) content assay and cell counting
kit-8 (CCK-8) with 4T1 cell
lines for protein activity evaluation.

I.
**Construction
recombinant plasmids**

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