PCR of TFAM mRNA

There are four types of mRNA that can be transcribed by TFAM DNA: TFAM-201 mRNA, TFAM-202 mRNA, TFAM-203 mRNA, and TFAM-204 mRNA. Among them, TFAM-204 mRNA was used as it is the major mRNA that produces the longest amino acid-sequence (246 aa). Among 246 amino acid-sequences, the sequence that produces the first to 43rd amino acids is translated into the Mitochondrial Signal Peptide (MTS) that helps TFAM protein to travel from the cytosol the into mitochondria in the cell. However, in our experiment, TFAM will be attached directly to the data-stored DNA, so the amino acids are cleaved. Regarding the amino acid sequence, the 43rd to 50th amino acids act as a linker, the 50th to 122nd amino acids are translated into the HMG BOX-A, the 122nd to 152nd amino acids also act as a linker, the 152nd to 223rd amino acids are translated into the HBG BOX-B, and the 223rd to 246th amino acids act as a tail.

[Figure 1] : Different parts of the amino acid sequence in TFAM

The TFAM cDNAs from different cell origins, A172 (brain), MCF7 (breast), MKN45 (stomach), and A549 (lung), were tested via PCR and put into an agarose gel to test which cell has the gene to produce TFAM protein the most. The TFAM-204 mRNA coding sequence (CDS) was multiplied via PCR. The solution included distilled H2O, forward primer, reverse primer, TFAM-204 cDNA, the mixture of optimized DNA polymerase, dNTP, and the buffer. For an hour and a half, denaturation, annealing, and elongation were repeated to amplify TFAM-204 cDNA. After the PCR, the solutions were put into an agarose gel, where the protein was expressed.

The results of the agarose gel showed that human lung cDNA expressed the most TFAM-204 protein, indicated by the width of the bend with 636 base pairs; thus, human lung cells were used throughout the experiment. The results also implied that all the TFAM cDNA was amplified without errors.

pET vector digestion, ligation and transfection

pET28 vector and TFAM cDNA were then digested by BamH1 and Xhol. TFAM cDNA was inserted into the pET28 vector, and the pET28-TFAM vector was transformed into BL21 (DE3) E. coli strain. After harvesting the E. coli, the IPTG induction test was conducted to check if IPTG serves its purpose in removing the lac repressor, which interferes TFAM protein from being translated. Using SDS-PAGE gel, the differences were found between the E. coli genome with and without the IPTG. The E. coli genome with IPTG was able to produce the TFAM, and, thus, moved less than the lighter solution without IPTG and TFAM in the gel. This led to the conclusion that IPTG is able to serve its goal in supporting T7 polymerase from expressing the target gene.

E. coli Lysate Purification

The E. coli lysate that contains the TFAM protein was collected to be purified. It was purified using the property that a nickel column, HIS-TAG on the end of the TFAM protein, can be attracted to the Ni-NTA magnetic nanobeads. The protein was purified by binding the protein to the magnet, washing magnetic nanobeads, and eluting target proteins. After the purification, using the SDS-PAGE Gel, protein was analyzed whether the purification process was effective after the purification. If the TFAM is not fully purified, in other words if it is still binded with other materials, the bend will move slower than the expected TFAM size, 28kDa, and thus, will be higher than the expected TFAM. The results showed that all the TFAM proteins were 28 kDa, proving the effectiveness of the purification process.

Bradford Assay

After checking the degree of purification, the amount of TFAM protein produced (concentration) was tested via Bradford Assay. Using microspectroscopy, the absorbance of Coomassie Brillian Blue G-250 (CBBG) to TFAM was measured to check how much TFM was produced. The TFAM absorbance was then plugged into Beer’s Law to quantify the protein in the solution. The calculation showed that the TFAM protein concentration was 0.235 µg/µL and 8.38 µM as a mol concentration.

TFAM-DNA Binding Test

To test the optimal mol ratio between the TFAM and the DNA, the TFAM-DNA Binding Test was conducted. As more TFAM binds with the DNA, the DNA will be heavier, and thus, will be slower than less TFAM binding DNA. Solutions with the same amount of DNA but with different amounts of TFAM were placed into the gel to be compared. The total volume of each solution was kept the same by buffers.

As the placement of the bend differed by the concentration of TFAM, it was concluded that the TFAM is able to form TFAM-DNA complex even in in-vitro conditions. Moreover, as the bend placements didn’t differ much between () and (), it was also concluded that the most effective TFAM:pSmile (2087 bp) binding mol ratio is 115.19:1, similar to the known TFAM:DNA binding mol ratio, 113.47:1.

Stress Test

To test whether TFAM protein can effectively protect data-stored DNA from various damages like UV light and H2O2, each TFAM-DNA complex was exposed to UV irradiation and H2O2 for 5 hours. After applying UV and H2O2 stress, Sanger sequencing was performed to determine the nucleotide sequence in the DNA. Then, the sequence was converted into binary code and was decoded back to the original binary image form.

[Figure 3]: Evidence of DNA protection of TFAM against UV irradiation. Agarose gel electrophoresis of pBHA/smile (lane 1-2), and pBHA/smile + TFAM (lane 3-5) treated as described in the protocol. Lane 1 to 5 from left to right.

After forming the TFAM-DNA complex with the molar ratios 86.39 and 115.19 (lane 4-5) the majority of the DNA still remained even after the UV stress for five hours. However, a TFAM-DNA complex with a low molar ratio of TFAM (14.40) was insufficient to protect DNA from UV irradiation (lane 3).

Although TFAM was able to protect the DNA to a certain degree, the band intensity of both 10 or 20 g/ml of TFAM (lane 4-5) was relatively low compared to the intensity of naked pBHA/smile (lane 1). This result suggests that the TFAM-DNA complex protects the DNA from UV irradiation, but it is insufficient to protect 100% of the DNA molecules in the sample.

Naked pBHA/smile (lane 1) is completely disintegrated by aggressive H2O2 stress (lane 2).

[Figure 4]: Evidence of DNA protection by TFAM against H2O2 as a stress factor. Agarose gel electrophoresis of pBHA/smile (lane 1-2), and pBHA/smile + TFAM (lane 3-5) treated as described in the protocol. Lane 1 to 5 from left to right.

In the solution with the TFAM-DNA complex with the molar ratio of 86.39 and 115.19 (lane 4-5), the plasmid DNA remained even after the 3 mM H2O2 stress for five hours. However, a TFAM-DNA complex with a low molar ratio of TFAM (14.40) was insufficient to protect DNA from H2O2 stress (lane 3). Similar to UV stress data presented, the band intensity of either the molar ratio of 86.39 and 115.19 (lane 4-5) was relatively low compared to the band intensity of naked pBHA/smile (lane 1). Therefore, similar to the UV irradiation test, the result indicated that TFAM protects DNA from H2O2 stress but does not protect 100% of DNA molecules in the sample.

In both UV irradiation and H2O2 stress, pBHA/smile without TFAM showed low recovery rates: 9% and 11%, respectively (lane 2).

[Figure 5] : After DNA stress was induced, the DNA was sequenced and decoded to retrieve the original image. (A) UV irradiated DNA (B) H2O2 stressed DNA. The intactness of the DNA was measured by how much of the original image has been preserved after each stress has been induced. The percentage indicates the average recovery rate of the original image (n= 3).

pBHA/smile with 1 µg/mL TFAM also showed a low recovery rate in both UV irradiated and H2O2 stressed samples: 21 % and 13%, respectively (lane 3). However, even with the UV irradiation and H2O2 stress, pBHA/smile with 10 or 20 µg/ml TFAM recovered 100% of the original image (lane 4-5). This result indicated that the TFAM-DNA complex protected the DNA even in aggressive aqueous storage conditions. Also, non-biological information, the smiley face image, stored in plasmid DNA could be retrieved without any errors.