Part:BBa_K4305001
pET28a-TFAM
This is the final plasmid construct to express TFAM protein with 6 x histidine tag.
Literature Review & Experimental Results:
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 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 5030
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4964
Illegal BamHI site found at 5134
Illegal XhoI site found at 5758 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 5030
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 5030
Illegal NgoMIV site found at 137
Illegal NgoMIV site found at 3184
Illegal NgoMIV site found at 3344
Illegal NgoMIV site found at 4932 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2263
References:
[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).
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