1st Lab Meeting

All wet-lab participants utilized a micropipette throughout all experimental procedures. Before the experiment, every student was instructed heavily and practiced the appropriate usage of the micropipette.

We have used six different micropipette volume types throughout the experimental procedure: P1000, P200, P100, P20, P10, and P2.

1. P1000 handles 1000 ~ 200 μL / Blue tip used
2. P200 handles 200 ~ 20 µL / Yellow tip used
3. P100 handles 100 ~ 10 μL / Yellow tip used
4. P20 handles 20 ~ 2 μL / Yellow tip used
5. P10 handles 10 ~ 1 μL / White tip used
6. P2 handles 0.5-2.5 µL / White tip used

[Figure 1]

1. Rotate rubber to set the volume to absorb.
2. Place the tip of the pipette into disposable tips.
3. Press the pressure (white) button with your thumb, for liquid absorption, before submerging.
4. Submerge the tip of the micropipette into the liquid. Press it until the first stop before uptaking the liquid.
5. Let the thumb go to absorb the liquid.
6. Stick the tip of the micropipette onto the wall of the tube.
7. Press the pressure button until the second stop to release all remaining chemicals into the container.
8. Press the second button next to the uppermost pressure button (used for absorption) to release the tip.

1. If the liquid being handled is minimal, skip #6 above.
2. Make sure to hold the pipette vertically with the tip down.
3. During absorption, submerge the tip just a little bit to yield more accurate quantitative results.
4. Change the tip every time the chemical changes to prevent potential chemical contamination.
5. Try to minimize waste as much as possible.
6. Red digits in micropipettes, if they have, indicate the decimal place. Make sure to be informed of how to measure the liquid volume since they all all of them differ by micropipette brands.

Four 4 types of mRNA can be transcribed by TFAM DNA: TFAM-201 mRNA, TFAM-202 mRNA, TFAM-203 mRNA, and TFAM-204 mRNA. Among them, we used TFAM-204 mRNA for our experiment.

TFAM-204 mRNA CDS (Protein Coding Sequence)

• 1 ~ 43 aa: Translates into Mitochondrial Signal Peptide (MTS) → Cleaved
  • MTS: Used only when TFAM protein travels from the cytosol into mitochondria in the cell.
  • We cleaved this particular coding sequence for our experiment because we will directly put the TFAM protein with the DNA.
• 43 ~ 50 aa: Linker
• 50 ~ 122 aa: Translates into HMG BOX-A
• 122 ~ 152 aa: Linker
• 152 ~ 223 aa: Translates into HBG BOX-B
• 223 ~ 246 aa: Tail

We conducted the PCR to multiply the TFAM-204 mRNA CDS that will be inserted into the vectors. The steps of the PCR are the following:

1. Denaturation: Heat 95℃ to separate DNA into a single strand.
2. Annealing: Cool the temperature to send DNA polymerase and several nucleotides toward DNA primer.
3. Elongation: Nucleotides form the copies of strands.
Procedural Material
1. F primer (Forward primer): 1 μL
2. R primer (Reverse primer): 1 μL
3. TFAM-204 cDNA: 1 μL
4. Water: 17 μL
5. Optimized DNA polymerase
6. dNTP
7. Buffer
Experiment Procedure

[Figure 2]

[Figure 3]
1. Mix 17.0 μL water, 1.0 μL forward primer, 1.0 μL reverse primer, and 1.0 μL cDNA inside the PCR tube.
2. Mix the liquid using the vortex.
3. Collect all the liquid on the tube wall inwards using the centrifuge.
4. Put the tubes in the PCR test machine (Thermal Cycler) and conduct the test for 1.5 hours.

[Figure 4]
Agenda 3: Agarose Gel Electrophoresis
Electrophoresis Principle
We conducted agarose gel electrophoresis to check if our TFAM-204 mRNA CDSs are duplicated well. The following is the principle of electrophoresis:
1. Electrical current (-) would push DNA (because DNA is generally negatively charged due to the presence of a phosphate group).
2. Small-sized DNA would move further than large-sized DNA.
Agarose Gel Explanation

Agarose is a small sugar-based molecule, and in agarose gel, agarose molecules are randomly distributed. We set the concentration of the gel as 1.5% because our DNA has about 600 bp.

Procedural Materials
1. 1.5 g Agarose powder
2. 100 ml TAE
3. Microwave heating (melting)
4. DNA staining solution (5 μL)
Experimental Procedure

[Figure 5]

[Figure 6]
1. Mix agarose powder with 100 mL TAE.
2. Mix 5 μL of DNA staining solution.
3. Heat in the microwave for 2 minutes.
4. Pour the heated liquid onto the gel caster and wait for 5 minutes.
   • Make sure that there are no bubbles when solidifying.
5. Put the agarose gel onto the gel tank.
6. Pour 400 mL of TAE buffer into the gel tank.
7. Drop 3 μL of the DNA size marker (used as the control group).
8. Drop 7 μL of the PCR testing sample.
Our Results

[Figure 7]

All TFAM DNA sizes were about 600 bp, as we expected.

2nd Lab Meeting

Agenda 1: PCR Solution Purification
Procedure Principle

This step is to purify the PCR solution by eliminating primer, DNA polymerase, and residues to only leave the pure amplified DNA.


[Figure 8]
Experimental Procedure
1. Add 75 μL of PB buffer and mix it with a vortex.
2. Transfer the mixture to the binding column.
3. Centrifuge for 1 min (balanced arrangement of the tubes when centrifuging).
4. Add 500 μL of NW buffer for washing.
5. Centrifuge for 1 min.
6. Discard flow-through.
7. Add 200 μL of NW buffer for washing.
8. Centrifuge for 2 min.
9. Place the column into the “new” 1.5 mL tube.
10. Add 40 μL of EB (elution buffer).
11. Incubation for 1 minute (so that more and more DNA can be dissolved).
12. Centrifuge for 1 min.
Agenda 2: Restriction Enzyme Digestion
Experimental Principle

Digestion of BamH1 & xho1 restriction enzymes onto TFAM DNA & pET28 vector DNA allows TFAM DNA insertion into the pET vector.


[Figure 9]
Procedural Material
DNA Solution:
• 1. DNA insert: 17 μL
• 2. 10X buffer: 2 μL
• 3. BamH1: 0.5 μL
• 4. Xho1: 0.5 μL
Vector Solution:
• 1. Vector: 17 μL
• 2. 10X buffer: 2 μL
• 3. BamH1: 0.5 μL
• 4. Xho1: 0.5 μL
Procedure

[Figure 10]
1. Mix 2 μL 10x buffer, 0.5 μL BamH1, 0.5 μL Xho1, and 75 μL PB buffer inside the digestion tube.
2. Vortex the mixture.
3. Incubate for 30 min
PCR purification
1. First, add 3 volumes of FB Buffer to the PCR product and then 1 volume of absolute isopropanol.
2. If the PCR product is 20 µl, add 60 µl of FB Buffer then 20 µl of absolute isopropanol. Mix them completely by vortexing. It is not necessary to remove mineral oil.
3. Transfer the mixture to the binding column and centrifuge the column at 14,000 rpm for 1 min. We recommend performing all centrifugation steps at room temperature.
4. Pour off the flow-through and re-assemble the binding column with the 2 ml collection tube.
5. Add 500 µl of W2 Buffer to the binding column and centrifuge at 14,000 rpm for 1 min.
6. Pour off the flow-through and re-assemble the binding column with the collection tube.
7. Repeat steps 4 and 5.
8. Centrifuge once more at 14,000 rpm for 1 min to completely remove residual washing buffer, and check that there is no droplet at the bottom of the binding column. Then, transfer the binding column to the new 1.5 ml microcentrifuge tube (not provided).
9. Add 30 µl of EA Buffer to the center of the binding column and wait for at least 1 min at room temperature for elution.
10. If DNA fragments are larger than 3 kb, increase incubation time to 10 min at 60°C. In the case of pure water, eluted fragment DNA may be denatured and unstable. EA Buffer, as well as TE buffer (pH 8.0) are suitable for ordinary downstream applications such as such as sequencing, restriction enzyme digestion, and ligation. On the contrary, the elution of fragment DNA with pure water may give the DNA denaturation and/or instability. TE buffer (pH 8.0) can be used as EA buffer, except that EDTA may interrupt the subsequent enzymatic reactions.
11. Elute the fragmented DNA by centrifugation at 14,000 rpm for 1 min.
12. If more DNA yield is required, elute the sample twice and use after concentration.

*Incubate the mixture solutions in the incubator for 10 minutes so that we provide enough time for enzymes to cut the gene.
*Do the second PCR purification to eliminate BamH1 & Xho1 restriction enzymes for ligation.

Agenda 3: Ligation
Experimental Principle

We inserted the TFAM DNA into the pET28 vector of E. coli, which can produce the TFAM proteins in the next experiment.

Procedural Material
1. 10x ligation buffer: 2 μL (shifting the solution into optimal condition for reaction with DNA ligase)
2. Digested vector: 7.5 μL
3. Digested DNA Insert: 10 μL
4. DNA ligase: 0.5 μL
Experimental Procedure
1. Mix 10x ligation buffer, digested vector, digested DNA Insert, and DNA ligase into a 1.5 mL tube. Vortex the solution.
2. Centrifuge for 1 minute.
3. Incubate at 15°C for 4 hours.

3rd Lab Meeting

Agenda 1: BL21 (DE3) Cell Harvest
Experimental Principle

The BL21 (DE3) cell harvest will allow the cells to be collected and duplicated through PCR. Cells were collected by the harvested E-coli sample solution.

Procedural Principle

Once the samples are collected from the harvested E. coli solution, samples go through centrifugation. Centrifugation allows the comparatively dense cells and bacteria to be separated from the solution and gain a cell palette. After centrifugation, PBS and SDS loading dye was added to allow the nucleus and the membrane to break. Sonification is done to support the breaking of the cell nucleus. After sonification, the tubes go through centrifugation to remove the possible leftover solution in the tube. This allows the removal of any possibility of leftover solution mixed with the cell/bacteria.

Experimental Procedure

[Figure 1]

[Figure 2]
1. Label two tubes, one with IPTG and one without IPTG. Samples labeled ‘Sample number + IPTG’ and ‘Sample number - IPTG’ are samples with and without IPTG, respectively.
2. Swirl the flask to mix the bacteria.
3. Take the sample and use the P1000 micropipette (set to 100) and the largest tip to collect 1 mL in the tube, minimizing the opening of the lid and the formation of any bubbles.
4. Collect 500 mL of the control sample without IPTG (Sample number - IPTG) by using a P1000 micropipette (set to 050) and the largest tip.
5. Centrifuge the samples, balanced within the centrifuge.
6. Add 160 µL PBS using the P200 micropipette with a maximum volume of 160 µL.
7. Absorb and release the solution 10 times to loosen the cells all clumped together due to centrifugation through pipetting.
8. Add 40 µL 5xSDS loading dye using the P200 micropipette with a maximum volume of 160 µL.
9. Absorb and release the sample a few times to make the solution even.
10. Sonicate samples with a total running time of 1 min per sample.
    1. Pulse on for 1 second and off for 10 seconds, repeated to prevent protein denaturation due to the sample heating.
    2. Power set to 1% because there was only a small amount of substance, and the power of the sonicator is too strong in comparison.
    3. Wipe the tip of the sonicator with alcohol after every repetition to prevent samples from being mixed.
11. Centrifuge the samples at 12000 RPM for 1 min.
Agenda 2: SDS-PAGE Gel
Experimental Principle
The SDS-PAGE gel was performed to identify the E.coli sample with the most TFAM protein.
Procedure Principle

The buffer solution was set to 95’C to denature the protein (TFAM) and compare the proteine sizes to choose a colony of E coli.

By providing electric current, smaller proteins will move faster (positioned lower) and larger proteins will move slower (positioned higher / above). Thick bands in the SDS-PAGE gel indicate larger amounts of proteins, while thinner bands indicate smaller amounts of proteins. TFAM protein with His tag has a size of approximately 20 kDa. Therefore, the band thickness was specifically focused on.

Experimental Procedure

[Figure 3]

[Figure 4]
1. Add buffer set to 95’C (50 mL buffer solution to 450 mL water).
2. Add standard protein marker to both ends in the gel.
3. Load all 8 samples into the gel.
4. Give electric current.
   1. 100V for one hour

4th Lab Meeting

Agenda 1: TFAM Protein Purification (Ni-NTA Magnetic Nanobeads)
Experimental Principle

The protein purification process will separate the desired protein (TFAM) from all other proteins present within E. coli.

Experimental Procedure

[Figure 5]

[Figure 6]
1. Preparation of E. coli lysate
   1. Prepare 3 mL of pre-induced bacterial culture with IPTG (Sample #2).
   2. Harvest the cells by centrifugation for 1 min at 12,000 rpm at 4℃ and discard the supernatant.
   3. Resuspend the cells in 500 µL of binding/washing buffer using a P1000 micropipette.
   4. Sonicate the cells using a sonicator (equipped with a micro-tip on ice).
      1. Pulse on: 3 sec / off: 10 sec
      2. Total running time: 3 min
      3. Power: 25%
      4. Repeat twice to make sure membranes are properly broken
   5. Centrifuge the E. coli lysate for 5 min at 12,000 rpm at 4℃ to pellet the cell debris & save the supernatant.
   6. Take 10 µL of the supernatant (lysate sample) for SDS-PAGE analysis using a P20 micropipette.
2. Equilibrating Ni-NTA magnetic nanobeads
   1. Transfer 1 mL of Ni-NTA magnetic nanobeads to a 1.5 mL tube & place the tube on a Neodymium (Nd) magnet for 1 min using a P1000 micropipette (set to 1 mL).
   2. Remove supernatant using P1000 micropipette.
   3. Equilibrate by adding 1 mL of binding/washing buffer to the bead slurry & mix briefly using a P1000 micropipette.
   4. Place the tube on Nd magnet for 1 min & remove supernatant.
   5. Repeat steps c & d once more.
3. Protein binding
   1. Load up to 500 µL of cleared lysate onto pre-equilibrated magnetic nanobeads using a P1000 micropipette.
   2. Mix by inverting 5-10 times.
   3. Place the tube on the Nd magnet for 1 min & remove supernatant (unbound sample).
4. Washing magnetic nanobeads (to remove E. coli proteins that are still present through nonspecific binding)
   1. Add 1 mL of binding/washing buffer & wash magnetic nanobeads by gently pipetting,
   2. Place the tube on Nd magnet for 1 min & remove supernatant (washing sample),
   3. Repeat a & b three times.
5. Eluting target proteins
   1. Add 200 µL of elution buffer & gently mix (to elute target proteins from magnetic nanobeads).
   2. Incubate for 1 min at room temperature.
   3. Place the tube on the Nd magnet for 1 min & collect supernatant (elution sample).
   4. Repeat elution step (a) once more.
   5. Identify purification steps by analyzing each sample with SDS-PAGE.
Agenda 2: SDS-PAGE Gel
Experimental Principle

The SDS-PAGE gel was performed to ensure that the TFAM protein was properly purified and separated from all other proteins.

Experimental Procedure
1. Load all samples from the TFAM protein purification process.
2. Add a standard protein marker to one end.
3. Give electric current.
   1. 100V for 30 minutes
   2. 120V for 3 hours
Experimental Results

TFAM protein was successfully purified.

5th Lab Meeting

Agenda 1: Bradford Assay (Protein Quantification)
Materials Needed
1. Bradford reagent (Bio-rad)
2. Bovine serum albumin (BSA) (2 mg/mL)
3. 96 well plate
4. UV/vis spectrometer
5. Distilled water
6. 1.5 mL, 2 mL tube
Experimental Principle

Bradford assays are used to perform a quantitative analysis of the unknown protein. Comassie Brillian Blue G-250 (CBBG), an absorbing medium, reacts with the protein, TFAM. Once the reaction finishes, the protein changes from maroon to blue: as more protein reacts with CBBG, the blue becomes darker. Once the reaction is over, using microspectroscopy, the absorbance of Comassie Brillian Blue G-250 (CBBG) to protein (in this case, TFAM) can be measured. The data then can be plugged into the equation from Beer’s Law to quantify the protein in the solution.


[Figure 6]
Experimental Procedure

[Figure 1]

[Figure 2]
1. In the 1.5 mL tube, mix the BSA and distilled water to make a BSA standard curve sample and calculate the density of the solution.
   
2. Use the 2 mL tube to dilute the Bradford reagent by mixing water (by making the ratio 1:5) for the unknown sample.
3. Add 195 µL of the diluted Bradford reagent to the 96 well for each sample, a total of 7.
4. Add 5 µL of BSA standard sample curve and the unknown sample to match a total volume of 200 µL.
5. Mix the sample with the reagent using a pipette, minimizing the air bubbles being created in the sample.
   
6. Wait for five minutes.
7. Use a microplate reader to record the absorbance at a wavelength of 595 nm.
Agenda 2: TFAM-DNA Binding Test
Experimental Principles

Agarose is a small sugar-based molecule, and in the gel, agarose molecules are randomly distributed. Agarose gel was used to test the binding of TFAM to the pSmile vector (DNA). The electrical current (-) would push the DNA because DNA is generally negatively charged due to the presence of a phosphate group, and small-sized DNA would move further than the larger ones due to its mass. As more proteins are present, the bends get thicker when the gel is loaded. Through this experiment, we’re able to compare the size and amount of protein in different samples. As the pSmile vector binds with the TFAM, the sample will get heavier and larger. Thus, as more TFAM is added, the sample would move at a slower rate.

Experiment Procedure

[Figure 3]
1. Prepare nine tubes.
2. Put 3 µL of DNA to each test tube.
3. Starting from 0 µL, increase 1 µL of TFAM per test tube so that the 9th test tube contains 8 µL of TFAM.
4. Starting from 8 µL, decrease µL of elution buffer being added to the test tube so that the total volume of solution is set to 11µL.
5. Add 2 µL of DNA loading dye at the end to load the gel.

6th Lab Meeting

Agenda 1: TFAM-DNA Stress Test
Procedural Principle
To test the stability of the TFAM-bound DNA, UV stress and H2O2 were added to the DNA solution.
Experimental Procedure

[Figure 4]
3M H2O2 Test
1. Prepare 2 different tubes, labeled ‘Elution, 3M H2O2’ and ‘TFAM, 3M H2O2.’
2. Add 3 µL of DNA, 8 µL of the elution buffer, 1 µL of 3M H2O2, and 2 µL of DNA loading dye to the tube labeled as ‘Elution, 3M H2O2.’
3. Add 3 µL of DNA, 8 µL of TFAM, 1 µL of 3M H2O2, and 2 µL of DNA loading dye to the tube labeeled as TFAM, 3M H2O2.’
4. Centrifuged the samples for 10 sec at 13000 rpm
5. Wait for 60 minutes.
6. Run the agarose gel electrophoresis



UV Test

[Figure 5]
1. Prepare 2 tubes, labeled ‘Elution, UV’ and ‘TFAM, UV.’
2. Add 3 µL of DNA, 8 µL of the elution buffer, and 2 µL of DNA loading dye.
3. Add 3 µL of DNA, 8 µL of TFAM, and 2 µL of DNA loading dye.