Experiments

Experiments Meetings

First Experiments Meeting

[Agenda 1: Pipette Practice]

Experimental Principle

Micropipette practice
- Before carrying out the PCR test, micropipette practice is required for better accuracy and precision.

Materials Needed

Micropipette: precise volume control range from 1000µL ~ 0.1µL
Used a small capacity pipette for our experiment (2, 10, 20 µL)

Procedure

Connect the pipette to the tip.
- The button on the side should be close to the hand.
Press the button on the top and release all the air inside.
- Push the button until the first stop.
Place the pipette inside the liquid.
- Make sure that the pipette is perpendicular to the liquid.
Slowly release the button and absorb the liquid.
Pour the liquid inside the pipette into a small container.
- Pour the liquid close to the container in contact.
- Pour the liquid onto the bottom middle of the container.

Figure 1 (Korea-HS team member pipetting with a small container, practicing for better accuracy and precision for the PCR test)

[Agenda 2: Polymerase Chain Reaction Test]

Experimental Principle

Polymerase Chain Reaction (PCR) Test
- The objective of the experiment is to check if the two proteins Afamin and Wnt3a are in the plasmid accurately or intact.
- In addition, the PCR test is done to rapidly produce tons of copies of a DNA segment between Primer F and Primer R.

Materials Needed

Primer F 1 µL
Primer R 1 µL
Plasmid DNA 1 µL
Water 17 µL
DNA Polymerase
Nucleotide (dNTP) - [ATGC]
Buffer: 5,6,7 are already placed in the tube

Procedure

Denaturation
- 95°C Single Strand for 1 minute.
Annealing Step
- Attach 60°C Primer for 1 minute.
Extension
- 72°C DNA synthesis for 1minute.
N*( 2) as many times as cycle number.
10*(2) as many as 35 ~ 40 times during the polymerase chain reaction.
- If there is a reaction after amplification, it indicates that the result is positive.
- If there is no response after amplification, it indicates that the result is negative.

Figure 2 (Korea-HS team member running a PCR test by setting up the conditions of the RT-PCR machine)

Second Experiments Meeting

[Agenda 3: Agarose Gel Electrophoresis]

Experimental Principle

Agarose Gel Electrophoresis
- Our team aimed to check the quantity and quality of the DNA throughout the Gel DNA Marker Test. We used an Agarose gel standard DNA marker to identify DNA size.
- It is possible to see the band size getting thinner from top to bottom, which means that the smaller the DNA, the thinner the mark goes down. To measure the DNA quantity, we examined the band thickness, which directly correlates to how much DNA is contained.

Materials Needed

Agarose Gel electrophoresis
1x TAE buffer: (Buffer with Tris Acetate EDTA)
- Tris-acetate is used to match the pH
Agarose Powder
- 1.5% concentrated Agarose gel
Gel tank

Procedure

Figure 3 (Procedure 4: Korea-HS team members inserting the afamin and wnt3a protein-DNA samples into the holes of the gel tank)

Figure 4 (Procedure 7: Korea-HS team members examining the standard DNA marker, checking for DNA quality and quantity)

Add the TAE Buffer and the agarose powder used at 70~80° Celsius to make them soluble enough and add DNA staining solution.
- DNA staining is essential because it lets us physically identify DNA when we reflect the UV light or phe.
Pour the gel into the plastic mold and shape them.
Put a plastic mold on top of the main gel caster and solidify the gel into cube shapes at 40° Celcius.
Put our samples carefully (using the pipette) into the holes of the gel tank.
- If the PCR test has been operated correctly - the results.
Place the gel mold into the gel tank, and fill the tank with the buffer.
- The gel tank sends electricity onto the mold.
- The pipette needs to go under the liquid (buffer) because the procedure is quite difficult to inject the protein inside the well plate.
The gel tank sends electricity from the top to the bottom of the gel tank, gathering all of the DNA at the bottom of the well.
Wait 20 minutes to check how far it moves in the standard DNA marker.
- The DNA bend can move farther down when we turn on electric chem.
- Main objective: To find DNA purity.

[Information to Help Data Analysis]

Figure 5 (Strap bands: a picture of a standard DNA marker)

Analysis method
- If the band is shown as one clear band the DNA is pure enough
- IPrimary non-specific binding may result in an unpredicted band size
PCR Troubleshooting method
- Multiple bands convey that we need to increase the temperature
- No bands convey that we need to decrease the temperature
Reason of error
- Even though the chemicals and apparatus used are the same, the result resolution may differ due to skill level disparities.

Third Experiments Meeting

[Agenda 4: Determine the optimal ratio BII-CMV-AfmW3A Vector (AW) to Super PiggyBac Transposase Expression Vector (T) for efficient transfection of HEK293 cells]

Experimental Principle

Determine the optimal ratio.
- In this experiment, our objective was to determine the optimum ratio between the AW vector plasmid and T plasmid that would enable the AW vector to integrate into the nuclear genome of the host cell.
- To achieve this efficiently and test for different ratios with a sufficient number of trials, we divided into four separate groups.

Materials Needed

BII-CMV-AfmW3A Vector (AW)
PiggyBac Transposase Expression Vector (T)
Lipofectamine 2000 Transfection Reagent (Thermofisher, CAT#11668027)
DMEM cell culture media (Gibco)

Procedure for Groups 1 and 2

Groups 1 and 2 conduct the experiment with a 1:2 ratio of AW Vector Plasmid and T Plasmid.

AW Vector Plasmid: 500 ng/µL (ratio: 1 = 1 µL) + T Plasmid: 500 ng/µL (ratio: 2 = 2.5 µL) + Liposome (8 µL)
- Total volume: 11.5 µL
Place 1 µL of AW vector plasmid in tube 1
Place 2.5 µL of T plasmid in the same tube → Place it at the bottom of the tube so it can be mixed with the previous fluid as well
Place 8 µL of Liposome in tube 1
Repeat procedures 2-4 for tube 2
Add 200 µL of cell culture media (DMEM) to both solutions
Incubate for 10 minutes to make the complex

Procedure for Groups 3 and 4

Groups 3 and 4 conducted the experiment with a 1:5 ratio of AW Vector Plasmid and T Plasmid.

AW Vector Plasmid: 500 ng/µL (ratio: 1 = 1 µL) + T Plasmid: 500 ng/µL (ratio: 2 = 5 µL) + Liposome (8 µL)
- Total volume: 14 µL
Place 1 µL of AW vector plasmid in tube 3
Place 5 µL of T plasmid in tube 3
Place 8 µL of Liposome in tube 3
Repeat procedures 2-4 for tube 4
Add 200 µL of cell culture media (DMEM) to both solutions
Incubate for 10 minutes to make the complex

[Agenda 5: Preparing HEK293 Cells for Transfection]

Experimental Principle

DNA plasmid transfection
- In this experiment, we completed DNA plasmid transfection. In our project, transfection is the process of adding Afamin and Wnt3a into a human embryonic kidney cell called HEK293.
- In order to facilitate this effectively, we added liposomes to allow the AW Vector Plasmid to enter the human cell. The liposome is crucial in this procedure, as the DNA cannot be inserted into the HEK293 cell without it.
- We used stable transfection, which involves the transposase in the T plasmid. When T plasmid is present, the DNA (AW Vector Plasmid) can enter the host cell’s nuclear genome. It is efficient as it can sustain long-term expression of a transgene by integrating foreign DNA into the host nuclear genome.

Materials

HEK293 cell line
DMEM culture media (Gibco)
Trypsin-EDTA cell dissociation buffer (Gibco)
Puromycin (10 mg/mL) stock solution (Sigma)
6 well-culture plate

Procedure

Use the aspirating machine to draw out base old media
Incubate the HEK293 cell with Trypsin-EDTA buffer to dissociate the HEK293 cells from the cell culture plate
Incubate the cell for 5 minutes in order for protease (Trypsin) to cut the membrane proteins from the cells
Condition 37 degrees Celsius and 5% CO2 level for mammalian cell culture
Incubate the cells with the prepared transfection complex for 6 hours
After aspirating the old media, add the fresh DMEM media to each sample
Add the puromycin in a final concentration of 10 µg/mL to select the HEK293 cells inserted with the Afamin and Wnt3a genes
Add the fresh DMEM media with puromycin (10 µg/mL) every three days
Culture the cells until the puromycin-resistant HEK293 cells are maintained

Fourth Experiments Meeting

[Agenda 6: Utilizing the Western Blot and Bradford Assay]

Experimental Principle

Western Blot
- The western blot can be used to determine the amount of Afamin and Wnt3a proteins produced in the cultured media.
  - This method will be performed by group 3.
Bradford Assay
- Before conducting the western blot, it is necessary to perform a Bradford assay to measure the total protein concentration. This will ensure that the same amount of protein is used during the western blot.

Materials Needed

Conditioned media samples from the previous experiment
- Contain HEK293 cells that include the vectors in the conditioned media
- Standard sample: sample #6
- Our sample: sample #7
Coomassie Brilliant Blue
- Dye used to determine the protein concentration for the Bradford assay
Graphing tool
- Used Google Sheets to graph
- Bovine serum albumin (BSA)

Procedure

Figure 6 (Table 1: Preparation of standard curve sample)

Bradford Assay
- The following condition was used to prepare the six samples of standard curve sample (Figure 6).
- Add 5 µL of standard samples to the well in the microplate
- Add 95 µL Bradford reagent to each well
- Mix the solution in the microplate wells gently by pipetting up and down (Figure 7)
  Figure 7 (Bradford assay standard sample using 94-well plates)
Prepare Sample Wells
- Take the protein samples (5 µL) and add them to the microplate.
- Add 95 µL Bradford reagent to each well
- Mix the solution in the microplate wells gently by pipetting up and down
- Allow the standard and sample wells in the microplate to incubate at room temperature for approximately 10 minutes. During this time, the Bradford reagent will bind to the proteins.
Measure Absorbance
- Set your microplate reader to measure absorbance at 595 nm.
- Zero the microplate reader using the blank well containing distilled water.
- Measure the absorbance of each standard solution and your protein samples in the microplate.
Calculate Protein Concentration
- Plot a standard curve using the known concentrations of the BSA standard and their corresponding absorbance values using a spreadsheet.
- Determine the absorbance of our protein samples.
- Using the standard curve, find the corresponding protein concentration for each sample based on their absorbance values.

Figure 8 (Korea-HS team member inserting each sample into the wells)

Figure 9 (Korea-HS team member inserting bovine serum (BSA) into the wells containing samples)

Figure 10 (Image of completely inserted samples and a mixture of BSA and samples. The wells written S0~5 row A and row E are where the standard samples were inserted into. The wells written 1~7 row C and row G are where our samples were inserted into.)

Fifth Experiments Meeting

[Agenda 7: Analyzing Our Western Blot Results]

Experimental Principle

Western Blot Results Analysis
- The goal of this experiment is to specifically look at the amount of Afamin and Wnt3a protein using the Western Blot.
- After going through this process, only the proteins with Afamin and Wnt3a will appear (chemiluminescence or HRP).
- Hence, after the utilization of Western Blot, it is possible to determine the optimal condition in which Afamin and Wnt3a are most produced as the greater number of Afamin of Wnt3a protein will indicate a brighter color.

Materials Needed

Protein Samples: Conditioned media samples were collected in previous experiments
Electrophoresis Equipment
- Gel electrophoresis apparatus (tank, power supply, gel trays, and combs)
- Polyacrylamide gel (usually SDS-PAGE gels) or precast gels
- Electrophoresis Buffers
- Running Buffer (Tris-glycine buffer)
- Protein Transfer
  - PVDF (polyvinylidene difluoride) membrane
  - Transfer apparatus (wet transfer systems)
- Blocking and Antibody Dilution
  - Blocking buffer (5% BSA in TBS)
  - Primary antibody diluent (usually the same as blocking buffer).
- Primary Antibodies: Afamin (Cat# AA 260-599) from antibodies-online and Wnt3a (Cat# 2391) from cell signaling targeting primary antibodies
- Secondary Antibodies: Horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit)
- Chemiluminescent Substrates
  - Chemiluminescent substrate reagents (e.g., ECL or SuperSignal)
  - Developer and fixer solutions
- Imaging Equipment
  - A chemiluminescence detection system (chemiluminescence imager)
Procedure
1. Gel Electrophoresis for Western Blot
  - Before the experiment, put the assigned loading buffer into each well
  - Well 1: DMEM-FBS (20mL)
  - Well 2: DMEM+FBS (20mL)
  - Well 3: No Treatment (20mL)
  - Well 4: Afamin & Wnt3a in 1:2.5 ratio (30mL)
  - Well 5: Afamin & Wnt3a in 1:5 ratio (30mL)
  - Well 6: Afamin & Wnt3a in 1:2.5 ratio (30mL)
  - Well 7: Afamin & Wnt3a in 1:5 ratio (30mL)
2. Heat the samples at 90 degrees for 5 minutes in an incubator as this process will denature the proteins inside.
3. Put in Tris-Glycine SDS buffer (running buffer) in the gel tank up to 2 gel labels to help electron glow in during electrophoresis.
4. Load the samples into an SDS-PAGE gel loading well using a micropipette (1 µg of total protein for the first trial and 10 µg of total protein for the second trial).
5. Put on the lid and set the gel electrophoresis tank to 120V
  - Transfer the proteins into the PVDF membrane using 100 V overnight.
  - Incubate Skim milk solution for blocking for 1 hour.
  - The primary antibody was incubated overnight
  - Using the PBST solution wash the PVDF membrane three times (For each washing - 5-minute incubation)
  - The secondary antibody was incubated for 1 hour
  - Using the PBST solution wash the PVDF membrane three times (For each washing - 5-minute incubation)
  - Chemidoc was used to detect the Afamin and Wnt3a protein band
6. Cell Proliferation Assay from Bovine-Cultured Cells
  - Detach the bovine cells attached to the bottom of the media container and put them in the well
  - Divide the wells into 7 groups for examination for a week (1 column for each day)
  - Use suction to remove the media from the now detached bovine cells
  - Use cell dissociation reagent and insert 1000 µL of it into the bovine sample
  - Incubate for 5 minutes
  - Put DMEM solution into the detached bovine cells 6 times with a 1000µL pipette and mix well
  - Use a 200 µL pipette and put 100 µL of the bovine cell and DMEM solution 7 times, one for each of the 7 well groups.
Figure 11 (Image of cultured bovine cells in an incubator observed by an electron microscope)

Figure 12 (Image of gel tank filled with Tris-Glycine SDS buffer (running buffer) and bovine cell culture sample of various concentrations (Well 1:12.1 µL, Well 2:10.1 µL, Well 3:9.7 µL Well 4:8.9 µL, Well 5:11.1 µL, Well 6:10.3 µL, Well 7:9.7 µL)

Figure 13 (Korea-HS team member inserts Tris-Glycine SDS buffer (running buffer) into the wells containing samples grown with Afamin and Wnt3a)

Sixth Experiments Meeting

[Agenda 8: Biochemical assay]

Experimental Principle
- Through this experiment, we wanted to see the effect of Afamin and Wnt3a protein on cell proliferation and compare that effect to the positive control which had FBS.
  - We measured this effect by adding PrestoBlue, a chemical reagent that allows us to perform a biochemical assay to examine the growth of the bovine cells.