Team:Shanghai High School/Experiments

The characterization protein and early diagnosis of cervical cancer

Here we demonstrate specific protein expression variations related to NFX1 over-expression, a gene found to be closely associated with cervical cancer.

Experiments

-Experiment and Results
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Lab preparation training
[• Preparation of lysogeny broth]
[Aim: Prepare for the incubation of E. coli]

We prepared 800mL liquid culture medium and each 100mL is put in one conical flask. We prepared 200mL solid culture medium also with each 100mL in one conical flask. Aluminum foil was used to cover the eight conical flasks and the flasks were sent to autoclave for sterilization (under 121°C) because our experiment involved incubation of E. coli, which required sterile conditions.

Components for preparing each broth:
- [1L liquid culture medium]: 10g tryptone, 10g NaCl, and 5g yeast extract

- [1L solid culture medium]: 10g tryptone, 10g NaCl, 5g yeast extract, 10-15 grams agar, and 1.47g 0.1M CaCl2.
Left Figure: liquid culture medium /Right Figure: solid culture medium

[Disinfect the Laboratory requirements]
[Aim: since our experiment and samples should be kept under the Sterile environment, all the apparatuses should be disinfected during the preparing stages.]

With the conical flasks, we also autoclaved following apparatuses:
- 3 boxes of 1.5mL EP tubes
- 2 boxes of glass cell spreaders
- 1 box of 50mL centrifuge tubes
- 1 box of 5mL tips
After being autoclaved, these were further sent to the drying machinery.

[• Preparation of competence cell medium]
[aim: to alter cell wall of cell rendering it easier to take in foreign DNA]

We prepared two reagent bottles. Add 1.47g calcium chloride into each of the two reagent bottles. In one of the bottle, 100mL water was added to dissolve calcium chloride; 70mL water and 30mL 15% glycerol for the other. We then closed the lids and shook the bottles to mix up the components added. After shaking, we slightly loosened the lids and covered the lids and bottlenecks with aluminum foil. We autoclaved the solutions and then cooled them to 40°C. Two bottles were stored under 4°C overnight.

[• Preparation of gel for protein electrophoresis (15% SDS-PAGE)]
[Aim: To identify the target proteins later in the experiments]

Synthesis of Protein electrophoresis gels:
- stacking gel
- 20mL stacking gel mix
- 200µL APS
- 10µL TEMED
- separating gel
- 45mL separating gel mix
- 400µL APS

- 15µL TEMED
The figure is the test of protein electrophoresis gel

We first set up gel cassettes and test with water whether their bottoms were well-sealed. Pipette 5mL separating gel into each cassette and wait for it to dry up. Then, fill the cassettes with stacking gel and insert the comb. After the stacking gel dried up, we stored the gel in packets for later use.

[• Picking mono-clone]
[Aim: Before the experiments start, the large amount of E.coli sample is needed to be prepared. In other words, high concentration of DH5α and BL21(DE3) are needed.]

The process was done at the clean bench working with two kinds of E. coli: DH5α and BL21(DE3). Use toothpick to transport monoclonal from petri dishes to tubes filled with lythogeny broth. Tubes were later put in shaking machine at 220rpm under 37°C overnight.

This Figure shows the Reproduction of DH5α and BL21(DE3) by shaking properly.
[Results & Conclusion:]
All the preparations for experiments are successfully completed. Therefore, our experiments are on the right track and ready to continue forward.
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[• Preparation of competent cells/Insert target gene sequences]
[Aim: By culturing two E. coli, the number of cells required for the experiment is reached, and the cell wall mechanism is weakened so that foreign DNA enters to allow it to be cultured in the cells. Through the cell carrier of E. coli, the desired specific gene chain is deliberately cultivated, and the desired cell structure is prepared for later experiments.]

(1) cultivating competent cells (precise data results from lab preparation)

DH5α (1)	DH5α (2)	BL21(DE3) (1)	BL21(DE3) (2)
0.660 abs	0.391 abs	0.731 abs	0.431 abs

We took out the tubes that were in shaking machine overnight. Respectively, we pipetted 1mL bacteria solution of DH5α into conical flasks with 100mL culture medium in it. We observed that BL21 bacteria grew rather vigorously, so we pipetted 1mL of it from tubes into 50mL culture medium. Use aluminum foil to tightly cover the opening of conical flasks and put the flasks in shaking machine for 2-3 hours. After that, pipette 5mL of bacteria solution out of each conical flask (4 in total) to test in spectrophotometer, which could reflect the extent of growth of bacteria.

(2) centrifuge
We precooked the centrifuge to 4°C and set it to 4000rpm for 10 minutes. We then took out the two precooked PC tubes and pipetted respectively 50mL of BL21 and DH5α into each of them. Start the centrifuge. When it stopped, pour out waste solution and repeat this process until all cells in the conical flasks were separated out in the PC tubes. Two PC tubes were resulted each with all DH5α cells and BL21 cells which were in the conical flasks.

(3) competent cell incubation
Following processes were done on ice. Add 5mL calcium chloride which contained 15% glycerol into each of the two PC tubes. Mix them evenly and place them in ice. We then pipetted each of the solution to every new EP tube by 50µL each time. After each pipetting, the tube was put immediately into liquid nitrogen. Later, these tubes were stored under -80°C. Took out 9 of them with DH5α and 1 with BL21 for
transformation.
[• PCR]
[Aim:PCR can rapidly amplify any known gene or DNA fragment of interest, thereby achieving a sufficient number of gene chains for experimental use. DNA electrophoresis is used to detect whether the PCR has amplified the correct gene sequence.]
(1) preparation of PCR component
We prepared 8 samples of PCR components with the same primer to react under different annealing temperatures to test the efficiency of annealing DNA chains under each condition.
- PCR component (20µL)
- primer (Yo-T7-pSB1C3-R &Yo-GFP-F): 0.8µL each
- template (pSB1C3-T7-B0034-GFP): 0.4µL
- 2× phanta buffer: 10µL
- dNTP: 0.4µL
- DNA polymerase: 0.4µL
- water: 8µL
(2) conducting PCR

We set 34 cycles and 95°C, 56°C, and 72°C correspondingly for denaturation, annealing, and extension. PCR products were prepared for competent cells. Then, DNA electrophoresis is operated in order to test if target gene is correct.
The figure above expresses the result of DNA electrophoresis under UV.

[• Transformation]
For each EP tube with prepared competent cells in it, we added 1µL DNA resulted from PCR. We cooled them on ice for 30 minutes and then carried out heat-shock under 42°C for 90 seconds. We added 300µL culture medium and placed them in shaking machine under 37°C at 220rpm for 45-60 minutes.

[• Plating E. coli]
We carried out this process at an E. coli clean bench. Centrifuge the culture medium after shaker so that the bacterial colony could precipitate. Remove part of the supernate and mix the rest of centrifuge products. Pipette   of the centrifuge products to petri dishes and use cell spreaders to spread the cells evenly across the dishes until the surface of the dishes dries up. The whole process was done under room temperature.

[Results & Conclusions:]
PCR products did not grow clones after transformation and the problem that we speculated would be Primers’ compatibility. Therefore, we decided to replace the primers and improve the method.

(• Run the gel for protein electrophoresis
We took out the gel we prepared yesterday and placed in electrophoresis cell with buffer added. We pipetted respectively 10µL and 20µL of positive control, mCherry plasmids, and GFP to the well. We set 80V and switched to 150V after 20 minutes, which took 1 hour in total. When it ended, we removed the glass and stacking gel. We added dye and placed the gel on horizontal shaker to visualize bands on the gel.)
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Insert T7 Promoter into the plasmid
[Aim: Replace the Primers and create recombinant DNA inserting T7 promoter into the plasmid]

[• PCR]
Step1: preparation of PCR component
We prepared 8 samples of PCR components with the same primer to react under different annealing temperatures to test the efficiency of annealing DNA chains under each condition.
- PCR component (50µL)
- primer: 2µL each
- template (pSB1C3-T7-B0034-GFP): 1µL
- 2× phanta buffer: 25µL
- dNTP: 1µL
- DNA polymerase: 1µL
- water: 18µL
Step2: conducting PCR
We set 34 cycles and 95°C, 50-60°C, and 72°C correspondingly for denaturation, annealing, and extension. PCR products were prepared for competent cells.

 

[• DNA electrophoresis]
Step1: preparation of 1% agarose gel (1g Agar + 100mL 1×buffer)
We added approximately 50mL loading buffer to dissolve Agar and microwaved the solution until it became colorless. We cooled the solution for around 5 minutes and added the rest of loading buffer. We poured the solution into gel tray and insert a comb. The gel was formed as it cooled down at room temperature, and comb was then removed.
Step2: run the gel

We mixed 5µL nucleic acid dye with 50µL of each PCR product. We insert each of the mixture in the well correspondingly. We ran the gel for 30 minutes at 110V and then observed under UV light.
Figure above shows the results of PCR under the UV
Step3: recycle the DNA
We carved out eight DNA fragments which were visualized by the bands under UV light and put them in EP tubes. Add 400µL binding buffer into each of the eight EP tubes and melt the mixture in them at 65°C for 10 minutes. We used micropipette to pipette each of the mixture into each of eight absorption columns and placed the columns in  eight new EP tubes. Centrifuge them at 12000rpm for 1 minute under room temperature. We removed the waste fluid in tubes and added 600µL DNA washing buffer. Centrifuge them again at 12000rpm for 1 minute under room temperature. We then removed the waste fluid and centrifuged for two minute and at 12000rpm. The columns were left standing for around 5 minutes to vaporize the alcohol inside. 30µL ddH2O was added to the absorption column and heated at 65°C to dissolve DNA. We then waited for 3 minutes for DNA to dissolve. We had a fourth centrifuge at 12000rpm for 1 minute under room temperature. The water in EP tubes was pipetted to absorption columns and repeat the centrifuge with same condition. Remove the absorption columns and the solution left in EP tubes were DNA samples that we aimed to recycled from electrophoresis.
(4) measure concentration of DNA
We utilized Nanodrop spectrophotometer to measure concentration of DNA