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.

Literature Review & Experimental Results:

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 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 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.

>pET-28 a (+) plasmid sequence

atccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgc tcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatctcagtggtggtggtggtggtgctc gagtgcggccgcaagcttgtcgacggagctcgaattcggatccgcgacccatttgctgtccaccagtcatgctagccata tggctgccgcgcggcaccaggccgctgctgtgatgatgatgatgatggctgctgcccatggtatatctccttcttaaagt taaacaaaattatttctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcg agatctcgatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgcc gacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccc cgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctac tactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgagatcccggacaccatcgaatggcgcaaaacctt tcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcg cagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaa aaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgct gattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaac tgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcg caacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgt tccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgg gcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctg cgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccat gtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatgg cgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgatacc gaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccg cttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccc tggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactg gaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccgggatctcgaccgatgcccttga gagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatc atgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgat cggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcg gcgagaagcaggccattatcgccggcatggcggccccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggcta ggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaac gtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccct gcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgc tggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaa ccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaa atcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagaca ttaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctga tgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgca gccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtg caccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctc actgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacaga atcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctgg cgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagga ctataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacct gtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttc gctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcc aacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgc tacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccag ttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaag cagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacga aaactcacgttaagggattttggtcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagc catattcaacgggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggc tcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaac atggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccg accatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggt attagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctg tttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgat gcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctc accggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattg atgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttca ttacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatga gtttttctaagaattaattcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcaca tttccccgaaaagtgccacctgaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctc attttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttc cagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggc ccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggag cccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgcta gggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcc cattcgcca

References:

[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).