Design

The purpose was to design a TFAM-DNA complex to enhance the stability of the DNA.

A549 (human lung adenocarcinoma cell line)

[Figure 1]: cDNA amplified from A172, MCF7, MKN45, and A549 human cell lines. The expected amplified TFAM (43 aa - 246 aa) is 636 bp.

The amplification of TFAM (43 aa - 246 aa) was performed using cDNA produced from A172, MCF7, MKN45, and A549 human cell lines. The amplified product from A549 showed a strong one band. Therefore, this amplified product was selected to be cloned into a pET28 expression vector (Figure 1).

The following primer sequences were used:
TFAM Forward primer containing BamHI site: 5' - ATAggatccatgtcatctgtcttggcaagt - 3'
TFAM Reverse primer containing XhoI site: 5' - ATActcgagttaacactcctcagcaccatattt - 3'

Build

A new DNA vector with the binary image data inserted was required for the experiment. In order to get the DNA vector, we have converted the image data into binary codes, and sent them to Bioneer for manipulation.

TFAM-204, the target protein used to create a TFAM-DNA complex, usually has 246 amino acid sequences. The first 42 amino acids function as a Mitochondrial Signal Peptide (MTS), which guides TFAM from the cytosol to the mitochondria. Since our project manually mixed TFAM with the DNA, the first 42 amino acids were unnecessary. To ease later experiments such as protein expression and complex creation, the unnecessary amino acid sequences were removed.

TFAM was also collected through a series of experiments. First of all, we manipulated a DNA vector and attached a DNA encoding protein to the vector.

[Figure 2]: Two ways of adding polyhistidine in the target gene. (A) The His-tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (B) The His-tag is added using primers containing the tag, after a PCR reaction the tag gets fused to the N-terminus of the gene.

We used method A to insert the TFAM gene into the pET28a (+) expression vector. The pET-28a (+) vector carry an N-terminal His•Tag®/thrombin/T7•Tag® configuration plus an optional C-terminal His•Tag sequence.

BamHI and XhoI restriction enzymes were used to cut both insert and vector.

Next, the DNA with TFAM from ligation was inserted into the E coli.

[Figure 4]: pET28-TFAM vector was transformed into BL21(DE3) E.coli strain.

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The protein was finally expressed through the pET expression system.

[Figure 5]: Principle of regulating gene expression using the pET-expression system.

[Figure 6]: The TFAM protein expression test was performed using four selected colonies with or without IPTG induction.

The expected TFAM protein size was 28 kDa, and the strong bands showed in colony # 2 and # 4 only in the IPTG-added sample. IPTG induces the inserted gene expression in pET28 containing E.coli (DE3) strain.

[Figure 7]: Protein purification using Ni-NTA magnetic silica resins.

The expression vectors contain an IPTG inducible promoter. The gene encoding a protein when cloned to an expression vector can be expressed by the induction using IPTG. The E. coli BL21 cells containing recombinant expression plasmid are grown to mid-exponential phase and then IPTG is added to the growing cells to induce the protein expression. The cell samples before and after the IPTG induction can then be analyzed for protein expression by SDS-PAGE analysis.

Colony # 1, Colony # 2, Colony # 4 samples were tested. Clear expected TFAM protein size bands were observed (Figure 7).

• We have performed SDS Page Gel to screen for the purification processes after experiments such as PCR and pET28 vector transfection.
• We have planned a Bradford Assay to quantify TFAM created during the experiment.
• We have planned a SDS-PAGE gel on solutions with different TFAM concentration to see if TFAM can bind with the DNA in lab conditions.

[Figure 8]: pET28-TFAM plasmid structure. The TFAM gene is cloned using BamHI and XhoI restriction enzymes.

Using the restriction enzyme BamHI and XhoI, the amplified TFAM gene was cloned into the pET28a expression vector. Figure 2 represents the final structure of pET28-TFAM.

Test

Agarose gel electrophoresis
Agarose gel electrophoresis was conducted to check if the correct protein, with a size of 28kDa, was amplified after PCR. Agarose powder and DNA staining solution were mixed with Tris-acetate EDTA (TAE) to produce the gel. Samples were dropped on different columns and electric currents ran through to analyze the samples. The more purified the protein is, the lighter it will be, thus the more it’ll move downwards.

Bradford Assay
Bradford Assay was used to quantify the amount of protein (TFAM) in the solution. Coomassie Brilliant Blue G-250 (CBBG), a dye, was used to stain the protein. Once the reaction finished, the protein changed from maroon to blue. Using microspectroscopy, the absorbance of CBBG to protein (in this case, TFAM) of the solution was measured. The data then was plugged into Beer’s Law to quantify the protein in the solution. The more TFAM there was in the solution, the more the color changed from maroon to blue.

[Figure 9]: Bradford assay was performed to measure the stock TFAM protein concentration.

The Bradford assay is a protein determination method that involves the binding of CoomassieBrilliant Blue G-250 dye to proteins. The dye exists in three forms: cationic (red), neutral (green), and anionic (blue). Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form (Amax = 470 nm). However, when the dye binds to protein, it is converted to a stable unprotonated blue form (Amax = 595 nm). It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer or microplate reader [2].

In any protein assay, the ideal protein to use as a standard is a purified preparation of the protein being assayed. In the absence of such an absolute reference protein, another protein must be selected as a relative standard. We used BSA standard solution to generate the standard curve (Figure 8). The calculated TFAM protein stock concentration is 0.235 µg/µL.

• Electrophoretic Mobility-Shift Assay (ESMA): The electrophoretic mobility shift assay was performed to determine if TFAM was able to form a complex with the DNA in a lab condition.

[Figure 10]: Electrophoretic mobility shift assay (EMSA) for TFAM binding into pBHA/smile. Band shift observed in DNA plasmid containing TFAM protein.

Naked pBHA/smile (lane 1) and pBHA/smile + BSA (lane 2) shows that most of the plasmid is in free DNA form as indicated in the gel (Figure 5). BSA is used as a negative control and shows that no DNA-BSA complex is formed. pBHA/smile with 20 µg/ml of TFAM (lane 3) band shifts upward indicating TFAM-DNA complex has been successfully formed.

• Bradford Assay
  • The absorbance measurements were arbitrary.
  • The absorbance measurements were converted to BSA standard concentration (µg/µL)
  • The Mol concentration was finally calculated based on the TFAM recombinant protein molecular weight.
• Plasmid DNA concentration measurements using nanodrop
  • The absorbance mesruements were arbitrary.
  • The equation (1) was used to calculate DNA concentration:
    
    
    C = concentration of neucleic acid in the sample
    A260 = The maximum absorbance measured by the nanodrop reading
    l = pathlengh of the nanodrop reading
    DF = dilution factor
    CF = conversion factor (50 µg/mL was used for double-stranded DNA)
    
    • The Mol concentration was finally calculated based on the plasmid DNA molecular weight.
• Agarose gel electrophoresis
  • The EMSA data was produced with relative units compared to the negative control sample (without TFAM protein)

Learn

[Figure 11]

As shown by the data retrieved from the DNA, TFAM:pSmile vector ratios of 86.39:1 and 115.19:1 were able to protect the data in the DNA from external stress factors, whereas other samples with low TFAM concentrations weren’t. This shows the effectiveness of TFAM in forming the TFAM-DNA complex and protecting the data in the DNA from extreme stress factors, which proves our hypothesis that a Transcription Factor A Mitochondria (TFAM)-DNA complex in an aqueous solution, where TFAM encapsulates the DNA to serve as a protection from various stress factors, could enhance the stability of DNA during storage and retrieval.

Reference

[1] Mierendorf, R C et al. “Expression and Purification of Recombinant Proteins Using the pET System.” Methods in molecular medicine vol. 13 (1998): 257-92. doi:10.1385/0-89603-485-2:257

[2] Kruger, N J. “The Bradford method for protein quantitation.” Methods in molecular biology (Clifton, N.J.) vol. 32 (1994): 9-15. doi:10.1385/0-89603-268-X:9