Cloning of Enhancer of Zeste Homolog 2 in Forward Orientation Into Escherichia Coli Using Histidine-Tagged Pbluescript Ii Ks+.

The EZH2 (enhance of zeste homolog2) is an enzyme that in humans is fixed by EZH2 and its supply the information’s about making of enzyme called a histone methyltransferase In this experiment PCR2 was examined whilst the EZH2 contributes to chemical modification. This resulted in repression. The aim of the study was to re-clone the EZH2 gene from 5’ to 3’ into another plasmid with pBluescript II KS fusing together with EZH2 moving forward. The histidine tags originate in the plasmid. Methods used encountered E.coli transformation, PCR, restriction enzymes, plasmid mini-preps and agarose gel electrophoresis. The end result lead to a 3’ to 5’ of EZH2 inserts. There was a good link which showed that the returning agar plate ensured that the collection of colonies consisted of forward oriented inserts.

Genetic engineering is the deliberate modification of the characteristics of an organism by manipulating its deoxyribonucleic acid (DNA). Bacteria possess plasmids which can be used as vectors for bacterial transformation. The plasmids can be altered to include donor DNA thus altering the physiological or physical characteristics of the bacterium. Escherichia coli have been found to be the most effective bacterium in genetic transformation and heterologous expression of human genes or proteins. It was hypothesised that Escherichia coli HB101 K12 could be transformed with a pGLO plasmid to express the genes encoded for green fluorescent protein (GFP), the bla gene for ampicillin resistance and the araC gene a regulatory protein in facilitating transcription and therefore the transformation efficiency for this bacteria could be calculated. Bacillus megaterium, E.coli B strain, Erwinia carotovora, Pseudomonas fluorescens, Staphylococcus epidermidis and Streptococcus lactis will also be subjected to transformation with the pGLO plasmid to collect transformation efficiency data for comparison against Escherichia coli HB101 K12. Escherichia coli HB101 K12 were found to have been successfully transformed with a transformation efficiency falling within the transformation protocol of 8.0 x 102 and 7.0 x 103. The incidence of Escherichia coli HB101 K12 on the LB/amp and LB/amp/ara pGLO+ plates indicated that the bacterial colonies present showed resistance to the ampicillin

Rarely, many other genes from the family members of ETS have also been identified as the fusion partners of EWS (7). According to Lin et al., 95% of cases of ES occur from EWS/FLI1 fusion gene formation as a result of transformation. Due to the difference in the locations of the EWS and FLI1 genomic breakpoints, it resulted in the existence of alternative forms of the chimeric gene. There are two most common forms, type 1 and type 2 accounting to 60% and 25% of the cases respectively. Type 1 consists of the first 7 exons of EWS joined to exons 6-9 of FLI1 and type 2 includes FLI1 exon 5 also.( 8) The prognosis of type 1 fusion is significantly better prognosis than the other fusion types as observed by Lin et al. (1999). A less active chimeric transcription factor is encoded by the type 1 fusion gene explaining the heterogeneous forms of ES at the molecular level. Particular chromosomal translocations is strongly associated with the development of PNET. The products of the fusion genes resulted from the translocations are specific to the type of tumors. In ES/PNETs, karyotype of t[11;22][q24;q12], which results from the EWS-FLII gene infusion and t[21;22][q22;q12], which results from EWS-ERG gene infusion account for 85% and 10% respectively (9-11). In reports of ES/PNETs, there are eight cases which chromosome translocation are t[11;22][q24; q12]

This technique was shown to provide “more effective control of locoregional recurrence in diseases like NSCLC as well as systemic control of micrometastases,” the spread of cancer cells where secondary tumors are too small to be detected (Roth, Swisher, & Meyn, 1999; “Micrometastases”, n.d.). As explained previously in this paper, p53 is involved in many pathways influencing the cell cycle and apoptosis, making it a good candidate for targeting using gene therapy. Moreover, although cancer cells have mutations in many genes that regulate growth and repair, because p53 plays such a significant role in apoptosis, the restoration of its function will induce apoptosis in the cancer

Two restriction enzymes, Nde1 and Xho1, enabled the gene coding for the H1a21c protein to be successfully inserted into the pET22b plasmid. However, since the pET22b vector contains a His- tag sequence, it was important that the Xho1 enzyme to introduce a stop codon that prevented a His- tag to be attached to the H1 protein.

A missing or defective p53 protein is usually the culprit in cancer, being that is in over 50% of cancers. The p53 gene codes for a protein that stops the growth as well as the development of tumors, as well as other functions. This is known as a tumor suppressor gene. If the gene is mutated, it allows damaged cells to survive and make tumors. The p53 gene can be mutated by cancer-causing substances in the environment. The p53 is responsible for proteins that fix damaged cells or cause damaged cells to die (apoptosis). It does this so the damaged cells do not pass on. The p53 gene prevents irregular cells from turning into tumors.

One the location of the gene is determined, the gene is then cloned (design primers and PCR the gene, insert gene into a vector). Cloning of the gene will allow for future experiments in which the gene can be manipulated in vitro or in

Since bacteria are haploid, asexually reproducing organisms it is important for these organisms to be able to accept genetic variability into their genome. A process called transformation, which involves absorbing small segments of DNA from deceased organisms in the natural world, does this. Transformation can also be mimicked in the laboratory using plasmid. Plasmids are small segments of DNA that occur in bacteria that allow us to regulate if transformation was successful. We attempted transformation of E. coli cells using plasmid called pVIB, which allows for luminescence and resistance to the antibiotic ampicillin, from Vibrio fischeri, however, we did not achieve a successful transformation.

The DNA fragment of PCMVCre-ERT was then inserted into the F1 zygotes to form a transgenic mice. PCR amplification was then performed using appropriate buffers, d NTPS, primers, DNA polymerase and the genomic DNA. For the analysis of the mouse F9 carcinoma cells, they were infected with pCre-ERT or pCre-ER. These cells were then cultured in the specific conditions and PCR amplification was carried out. Southern hybridization and PCR were used to detect the transgene and the trans allele. For the detection of the transgene by PCR, specific primers were used Cre-ERT and for the southern hybridisation, tail DNA digestion was performed. mRNA synthesis was then detected in the mice. RT-PCR was used for the estimation of the mRNA level. RNA isolation from the mouse was done using Licl/Urea method. Synthesis of cDNA and then amplification by 35 cycles of PCR resulted in 254 –bp fragment of cDNA. HPRT mRNA was used as the control. PCR and southern hybridisation were then again used for the analysis of Cre-ERT mediated excision of the marker from the allele. Specific primers were used for the amplification of wild type allele and the target allele. Southern blotting was then performed. Cre –ERT mediated gene excision was then estimated using semi-quantitative PCR and Cre –ERT mediated gene expression was estimated using semi quantitative PCR. Different number of amplification cycles were used for each samples. Agarose gel

(2A) Discuss how you would molecularly clone this gene into a vector by traditional recombinant DNA cloning methods, PCR cloning, or recombineering / synthetic DNA approaches.

The central DNA binding domain is the most highly conserved region of p53, when compared to its other family members, p63 and p73. Loss of tumor suppressor function of p53, as seen in most cancers, results from missense mutations in the DNA

P53 tumor suppressor plays a vital role in the prevention of transformation of oncogene. However, stresses from area such as DNA damage, oncogenes, spindle damages etc. can cause a mutation in TP53 tumor suppressor gene which can alter and cause disturbance in the P53 pathway which involved certain protein and genes regulator dealing with growth arrest, apoptosis, DNA repair and angiogenesis.

Gene cloning is a commonly used in a biological technique in which a gene is fused into a self-replicating genetic element known as a plasmid. When introduced into a host most likely bacterium, it will self-replicate and generate into a big number of identical copies of a certain type of gene. In a standard cloning experiment, the DNA that contains the desired gene is removed from the organism. Afterwards, the DNA is then cut into the correct size using restriction of enzymes. By using the same restriction of enzymes that's how the plasmid is treated. Using the same restriction enzymes to cut the DNA fragment clarifies that the ends of the vector fragment will match with the ends of the DNA fragment that needs to be cloned. The vector and DNA fragments are combined together and then they are to ligate together. Ligation is performed using ligase enzymes. The recombinant plasmid, has an insert that contains

The PCR cycle continues as DNA is copied exponentially. The original PCR methods allowed detection of DNA growth at the end of the process; however the introduction Real Time-PCR (RT-PCR) has allowed DNA amplification progress to be monitored throughout the assay (6).

DNA codes for protein sequences. Proteins are crucial in all cellular activities and govern the phenotypes of organisms (Campbell et al., 2014). New techniques have been developed in which mutations can be induced in specific loci within DNA, this process is known as Gene editing. DNA is removed, replaced or edited using a type of enzyme called nucleases. The nuclease creates a break at specific locations in DNA and utilises the cell’s biological machinery to repair the break incorporating new sections or eliminating existing sections by either homologous recombination or non-homologous recombination. A major problem affecting the applications of such techniques is off-target activity. This is when the nuclease cuts at an un-specified loci because of similar homology to the target sequence and introduces a random mutation (Gaj et al,. 2013). The