Part:BBa_K4305005

DNA sequence of the converted binary image of a smiley face (100bp)

A binary image (smiley face) is a digital image composed of 2 colors (black and white). Therefore, it is possible to represent a binary image as a binary code representing 0 = white and 1 = black. In this research, the smile face image was converted into binary code. Then, binary code was again converted into DNA code, where G or C represents binary code 0 and A or T represents binary code 1. Then, DNA was synthesized and cloned into pBHA vector (pBHA/smile).

Literature Review & Experimental Results:

The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology [1]. The technique for synthesizing DNA has developed, with the cost of DNA synthesis dropping annually. With this, the use of DNA as an information storage medium has been a notable idea. The process of DNA data storage proceeds as follows: a computer maps a string of bits (zeros and ones coding for a digital file) to sequences of DNA using so-called error correction codes [2]. Once a DNA sequence is constructed based on the digital file, DNA strands of the specific sequence can then be physically generated [3]. Currently, these generation DNA strands are commonly stored through freezing the DNA in solution, drying the DNA, or encapsulating the DNA molecules in small silica particles to shield the stored information from environmental factors [4]. The DNA sequences can be decoded back to the strings of bits, or the digital codes which make up the digital file, using methods such as Sanger sequencing. As data is stored in DNA, it is also possible for mass copies of the information using the polymerase chain reaction (PCR).

In this experiment, a binary image (smiley face) composed of two colors (black and white) was represented through nucleotide bases. The binary image was represented through a binary code with 0=white and 1=black. Then, this bimary code was again converted into DNA nucleotide bases where G or C represents binary code - and A or T represents binary code 1. Then, DNA was synthesized and cloned into pBHA vector (pBHA/smile).

Once this vector was formed, an electrophoretic mobility shift assay (EMSA) was carried out to identify the optimal mol ratio of TFAM to DNA complex. pSmile DNA bands were shifted to an upper position indicating that the TFAM-pSmile complex was successfully formed. The theoretical calculated mol ratio (TFAM:pSmile) was 113.47:1, and EMSA results showed maximum band shifts between mol ratios of 100.79 and 115.19, indicating that the maximum binding capacity of purified TFAM protein to pSmile DNA is between these mol ratios.

To test whether the TFAM protein can effectively protect data-stored DNA from various damages like UV light and hydrogen peroxide, each TFAM-DNA complex was exposed to UV irradiation and hydrogen peroxide for 5 hours. After applying these stress factors, 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. Naked pBHA/smile was completely disintegrated by an aggressive UV radiation. Additionally, it was shown that a TFAM-DNA complex with a low molar ratio of TFAM was insufficient to protect DNA from UV irradiation. The results as a whole suggested that the TFAM-DNA complex protects the DNA from UV irradiation, but is insufficient to protect 100% of the DNA molecules in the sample.

Naked pBHA/smile was again completely disintegrated by aggressive hydrogen peroxide stress. Again, a TFAM-DNA complex with a low molar ratio of TFAM was insufficient to protect DNA from hydrogen peroxide stress. Similar to the UV stress data presented, the band intensity of either the molar ratio of 86.39 and 115.19 was relatively low compared to the band intensity of naked pBHA/smile. Therefore, similar to the UV irradiation test, the result indicated that TFAM protects DNA from hydrogen peroxide stress but does not protect 100% of DNA molecules in the sample.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 38
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References:

[1] Hughes, Randall A. and Ellington, Andrew D. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 9, a023812 (2017).

[2] Meiser, Linda C. et al. Synthetic DNA applications in information technology. Nature Communications. 13, 352 (2022).

[3] Kosuri, Sriram and Church George M. Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. 11, 499-507 (2014).

[4] Paunescu, Daniela et al. Reversible DNA encapsulation in silica to produce ROS-resistant and heat-resistant syntehtic DNA 'fossils'. Nat Protoc. 8, 2440-8 (2013).