pET28a (+) Plasmid Vector - Part: BBa_K4305000

The pET-28a-c(+) vectors carry an N-terminal His•Tag®/thrombin/T7•Tag® configuration plus an optional C-terminal His•Tag sequence.

The pET series of expression plasmids are widely used for recombinant protein production in E. coli. pET28a is the most popular expression plasmid, containing the T7 promoter and an adjacent lac operator sequence induced to suppress uninduced expression [1]. Also known as Bacterial Recombinant Protein Vector, it is an expression vector used for the expression of recombinant protein in E.coli, using the T7 lac promoter system for controlled gene expression. The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E.coli target genes. These are cloned in pET plasmids under control of strong bacteriophage T7 transcription and, optionally translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that almost all of the cell's resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction [2].

Recombinant DNA technology (or gene cloning) refers to the transfer of a DNA fragment from one organism to a self-replicating genetic element such as an expression vector. The inserted DNA can then be propagated in foreign host cells or may be expressed to produce a recombinant protein [3]. Prokaryotic cells such as E.coli are the preferred host for the expression of foreign proteins because of their inexpensive, rapid biomass accumulation and simple process scale up [4]. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability due to the production of proteins potentially toxic to the host cell. Genes that encode a protein of interest are usually inserted into a restriction-enzyme based multiple cloning region downstream of a T7 promoter for IPTG-inducible transcription by the T7 RNA polymerase [5].

Experimentally, this pET28a plasmid vector was used to express the TFAM protein with 6x histidine tag. Within the experiment, the pET28 vector and TFAM cDNA were digested by BamH1 and Xhol. The TFAM cDNA was inserted into the pET 28 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 with 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 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.

The E.coli lysate that contained the TFAM protein was purified and its concentration was also tested via Bradford Assay. The optimal mol ratio between the TFAM and DNA was determined through the TFAM-DNA Binding Test. 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 with the total volume of each solution being 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 in in-vitro conditions. It was also concluded that the most effective binding mol ratio is 115.19:1, which is similar to the known binding mol ratio which is 113.47:1. This TFAM-DNA complex was then exposed to UV irradiation and hydrogen peroxide to test whether TFAM protein can effectively protect data-stored DNA. The TFAM-DNA complex with the optimal molar ratio showed that the majority of the DNA remained after being exposed to UV stress for five hours. Additionally, plasmid DNA remained even after the 3mM hydrogen peroxide stress for five hours as well, showing that the complex is sufficient to protect data-stored DNA.

The pET28 vector played an important role in this experiment to express TFAM protein with the 6x histidine tag in order to further utilize the TFAM protein that was expressed.

[1] Dubendorff, J. W. & Studier, F. W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59 (1991).

[2] Mierendorf, R. C. et al. Expression and Purification of Recombinant Proteins Using the pET System. Methods Mol Med. 13, 257-92 (1998).

[3] Liu, Zhi-Quiang and Yang, Ping-Chang. Construction of pET-32a(+) Vector for Protein Expression and Purification. N Am J Med Sci. 12, 651-655 (2012).

[4] Sahdev, Sudhir et al. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. 307, 249-64 (2008).

[5] Gay, Glen et al. Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae. Plasmid. 76, 66-71 (2014).

pET28a-TFAM - Part: BBa_K4305001

This is the final plasmid construct to express TFAM protein with 6 x histidine tag.

The use of pET28a-TFAM was used in multiple previous experiments similar to the procedure used in order to construct a TFAM-DNA complex to store data. The human TFAM gene has been cloned into the pET28a expression vector, forming a pET28a-TFAM. This construct encodes residues 43-246, corresponding to full-length TFAM after cleavage of the N-terminal mitochondrial leader sequence (residues 1-42). These TFAM mutants were constructed by PCR using oligonucleotides encoding mutations [1].

In other experiments as well, TFAM coding sequence without mitochondrial targeting sequence (residues from 43 to 246) has been cloned in vector pET28 (pET28-TFAM) by using NcoI and XhoI restriction sites, and this vector is Kanamycin resistant. In this experiment conducted by Cuppari, the TFAM expression protocol started with the transformation of BL21-DE3 pLys-S E.coli expression strain with pET28-TFAM [2].

The pET28-TFAM plasmid was therefore constructed in order to finally express the TFAM protein in an experiment similar to ours. Other experiments also involve pET28-TFAM in order to express other proteins such as HheD2, halohydrin dehalogenases D2 from Gammaproteobacterium. In this experiment, the plasmid which contained HheD2 gene was introduced into BL21-DE3 pLys-S E.coli expression strain with pET28-TFAM [3]. Undergoing further processes such as incubation, heat-shock step, and being incubated, the process was led to express the HheD2 protein that was needed in the experiment.

Experimentally, the pET28-TFAM plasmid is used to express proteins, which in our experiment was TFAM protein with 6 x histidine tag. In our experiment, the pET28-TFAM vector was transformed into BL21 (DE3) E.coli strain as well. 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 IPTG. The E.coli genome with IPTG was able to produce the TFAM, and, thus, moved less in comparison to the lighter solution without IPTG and TFAM in the gel. This led to the conclusion within the experiment that IPTG is able to serve its goal in supporting T7 polymerase in the pET28a (+) vector from expressing the target gene.

The E.coli lysate that contained the TFAM protein was purified and its concentration was also tested via Bradford Assay. The optimal mol ratio between the TFAM and DNA was determined through the TFAM-DNA Binding Test. 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 with the total volume of each solution being 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 in in-vitro conditions. It was also concluded that the most effective binding mol ratio is 115.19:1, which is similar to the known binding mol ratio which is 113.47:1. This TFAM-DNA complex was then exposed to UV irradiation and hydrogen peroxide to test whether TFAM protein can effectively protect data-stored DNA. The TFAM-DNA complex with the optimal molar ratio showed that the majority of the DNA remained after being exposed to UV stress for five hours. Additionally, plasmid DNA remained even after the 3mM hydrogen peroxide stress for five hours as well, showing that the complex is sufficient to protect data-stored DNA.

The pET28 vector played an important role in this experiment to express TFAM protein with the 6x histidine tag in order to further utilize the TFAM protein that was expressed.

[1] B. Ngo, Huu et al. Tfam, a mitochondrial transcription and packaging factor, imposes a U-turn on mitochondrial DNA. Nat Struct Mol Biol. 18, 1290-1296 (2011).

[2] Cuppari, Anna. Structure and biophysical studies of mitochondrial Transcription Factor A in complex with DNA. Universitat de Barcelona (2016).

[3] Petrillo, Giovanna. Insight into the structure and function of engineered biocatalysts: serine hydroxymethyltransferase from Streptococcus thermophilus and halohydrine dehalogenase D2 from Gammaproteobacterium. Universitat de Barcelone (2017).

TFAM (43aa-246aa) - Part: BBa_K4305002

The mature TFAM protein contains two HMG boxes separated by a linker and a charged C-terminal tail. HMG-box domains allow TFAM to bind, wrap, and bend DNA without any sequence specificity. C-terminal tail is required for activation of promoter-specific mtDNA transcription. TFAM gene (43aa-246aa) was cloned into pET28 vector to overexpress TFAM protein in E.coli. This vector allows the TFAM protein to be fused with a 6 x Histidine tag at the N-terminal region.

In contrast to the chromatin-based packaging of the nuclear genome, the mitochondrial genome is packaged into non-chromatin nucleoids involving proteins specific to mitochondria, such as TFAM [1]. TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)-the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1) [2].

TFAM functions in determining the abundance of the mitochondrial genome by regulating packaging, stability, and replication [3]. Human TFAM has an ability to bind to DNA in a sequence-independent manner and is abundant enough to cover whole region of mitochondrial DNA, owing to which TFAM stabilizes mitochondrial DNA through formation of the nucleoid and regulates (or titrates) the amount of mitochondrial DNA [4]. TFAM contains two high mobility group (HMG)-box domains (HMG-box A and HMG-box B) that intercalate into the minor groove of a half-site [2].

The mitochondrial genome contains three promoters - the light strand promoter (LSP), the heavy strand promoter 1 (HSP1), and the heavy strand promoter 2 (HSP2) [4]. When TFAM is bound to these promoters, either LSP or HSP, the TFAM protein distorts mitochondrial DNA into a U-turn [5]. TFAM forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. This is shown as each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of DNA, with the positively charged a-helix serving as a platform to facilitate DNA bending [4].

The mitochondrial protein is processed in six steps: (1) The protein containing the signal sequence is synthesized in the cytoplasm. (2) Signal sequence binds to a receptor in the organelle membrane. (3) Receptor - protein complex diffuses within the membrane to a contact site. (4) Protein is unfolded, moved across the membrane, and refolded. These operations are carried out by the protein transporter complex and its associated chaperone proteins. (5) Once inside, the signal sequence is cleaved off by a specific peptidase. (6) Finally, mature protein is formed inside the mitochondrial matrix.

In the experiment, 43-246aa TFAM was cloned into pET28 (N-terminal Histidine tag, E.coli expression vector) to produce His-tagged TFAM protein. The first 42 amino acids of TFAM guides TFAM into the mitochondria. We cloned the TFAM (43aa to 246aa), which does not contain the mitochondrial signal peptide because TFAM recombinant protein does not have to transport to mitochondria in this project. The mature TFAM protein contains two HMG boxes separated by a linker and a charged C-terminal tail.

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. HMG-box domains allow TFAM to bind, wrap, and bend DNA without any sequence specificity.

C-terminal tail is required for activation of promoter-specific mtDNA transcription. TFAM gene (43aa-246aa) was cloned into pET28 vector to overexpress TFAM protein in E.coli. This vector allows the TFAM-protein to be fused with 6 x Histidine tag at the N-terminal region.

While TFAM protein distorts mitochondrial DNA into a U-turn, in the TFAM-DNA complex, the DNA was not distorted. This is because TFAM and DNA are not bound at a specific promoter but they combine non-specifically, which therefore leads to a U-turn not being formed. Instead, TFAM and DNA were compactly binded to form the TFAM-DNA complex.

[1] B. Ngo, Huu et al. The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA. Nature Structural & Molecular Biology. 18, 1290-1296 (2011).

[2] B.Ngo, Huu et al. Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation. Nature Communications. 5, 3077 (2014).

[3] Kang, Inhae et al. The mitochondrial transcription factor TFAM in neurodegeneration: Emerging evidence and mechanisms. FEBS Lett. 5, 793-811 (2018).

[4] Kang, Dongchon et al. Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions. Mitochondrion. 7, 39-44 (2007).

[5] Rubio-Cosials, Anna et al. Protein Flexibility and Synergy of HMG Domains Underlie U-Turn Bending of DNA by TFAM in Solution. Biophysical Journal. 10, 2386-2396 (2018).

TFAM_43aa_BamHI_Forward Primer - Part: BBa_K4305003

This primer is used to amplify the TFAM gene (43 aa- 246aa) using human cDNA using PCR.

BamHI is a Type II restriction modification system from Bacillus amyloliquefaciens which recognizes the sequence GGATCC [1]. By recognizing this DNA sequence, it leaves an overhang of GATC which is compatible with many other enzymes. The BamHI-DNA complex is a sequence-specific endonucleases-DNA complex.

There are multiple experiments that have used BamHI sites on primers to overexpress or amplify a certain gene. In one experiment, primers targeting wild-type TFAM gene carrying BamHI (5'-CGAGGATCCACCATGGCGTTTCTCCGAAGC-3') and Notl (5'GTAGCGGCCGCATACACTCCTCAGCACCATA-3') restriction enzyme sequences were used to amplify full-length TFAM cDNA from SAS cells, which encode wild-type TFAM. This was ultimately used to construct the TFAM overexpressing vector [2]. In another experiment, a recombinant product of TFAM-GFP (green fluorescent protein) fusion was amplified using primers and cloned into a BamHI- and SalI- digest pG-1 vector [3].

A previous experiment was especially similar to our experiment, involving both TFAM protein and also pET28a vector. Mouse TFAM cDNA was amplified using the PCR primers and cloned into the Ndel and BamHI sites of pET28a. The resulting fusion protein excluded the N-terminal 32 amino acid mitochondrial localization sequence of TFAM, which was replaced with 23 amino acids containing a 6 x His tag and the thrombin cleavage site [4]. A sense primer that contains a BamHI site has also been used for amplification. In this specific experiment, the DNA fragments for human TFAM and the HA tag were digested with appropriate restriction enzymes and inserted between the BamHI and NheI sites of vector pTRE2hyg [5].

Experimentally, this forward primer was used to amplify the TFAM gene (43aa-246aa) using human cDNA using PCR. During this process TFAM cDNAs from different cell origins, A172 (brain), MCF7 (breast), and 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 water, 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 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 band with 636 base pairs; thus, human lung cells were used throughout the remainder of the experiment. The results also implied that all the TFAM cDNA was amplified without errors.

[1] Brooks, J.E et al. Cloning the BamHI restriction modification system. Nucleic Acids REs. 17, 979-997 (1989).

[2] Hsieh, Yi-Ta et al. Mitochondrial genome and its regulator TFAM modulates head and neck tumourigenesis through intracellular metabolic reprogramming and activation of oncogenic effectors. Cell Death & Disease. 12, 961 (2021).

[3] Schena, M et al. Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol. 194, 389-98 (1991).

[4] Brown, Timothy A. et al. Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA. Plos One (2016).

[5] Kanki, Tomotake et al. Architectural Role of Mitochondrial Transcription Factor A in Maintenance of Human Mitochondrial DNA. Molecular and Cellular Biology (2004).

TFAM_43aa_XhoI_Reverse Primer - Part: BBa_K4305004

This primer is used to amplify the TFAM gene (43 aa- 246aa) using human cDNA using PCR.

In order to clone human TFAM, XhoI sites have been previously used. Restriction sites were introduced by the forward and reverse (CCATCGATC CATTGTGAACACATCTC) primers. The TFAM insert in this experiment was subcloned by the XBaI and XhI sites to yield pBS-TFAM used for in vitro transcription / translation analyses. A truncated version of TFAM was produced lacking 25 amino acids at the C-terminal end, and the PCR product was cloned into the XbaI and XhoI sites using pBS-TFAM as a template and appropriate primers containing XbaI and XhoI restriction sites [1].

In other experiments, a similar process was repeated. Certain genes have been cloned into different expression vectors using XhoI restriction sites along with others such as NcoI and further expressed and purified specific proteins [2]. XhoI has also been used to digest fragments that are made. For example, an RBS-lambda phosphatase fragment was generated with primers and digested with KpnI and XhoI. This fragment was also ligated into the cloning vector which was previously linearized with BamHI and XhoI [3]. Polymerase Chain Reaction (PCR) is based on three steps required for any DNA synthesis reaction: (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers [4]. The XhoI reverse primer has been used in the second step, for annealing primers in order for genes to be synthesized based on the template strand.

Experimentally, the XhoI reverse primer was involved in the PCR reaction. 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 water, 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 band 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. Furthermore, the pET28a vector and TFAM cDNA were then digested by BamHI and XhoI. TFAM cDNA was inserted into the pET28 vector, and the pET28-TFAM vector was transformed into BL21 (DE3) E.coli strain.

[1] Garstka, Heike L. et al. Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA. Nucleic Acids Res. 31, 5039-5047 (2003).

[2] Litonin, Dmitry et al. Human Mitochondrial Transcription Revisited: ONLY TFAM AND TFB2M ARE REQUIRED FOR TRANSCRIPTION OF THE MITOCHONDRIAL GENES IN VITRO. Journal of Biological Chemistry. 285, 18129-18133 (2010).

[3] Burger, Michael et al. The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent. Advanced Science. 9, 2104987 (2022).

[4] Delidow, B.C. et al. Polymerase chain reaction : basic protocols. Methods Mol Biol. 15, 1-29 (1993).

DNA sequence of the converted binary image of a smiley face (100bp) - Part: BBa_K4305005

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 a pBHA vector (pBHA/smile).

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 binary 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.

[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).