From Gene to Protein, The Creation of Topoisomerase I
Summary
The production of topoisomerase I starts with pTrc99A and λZAPII/topAcysB, the templates of instruction for the enzymes molecular makeup. Incorporating the gene of interest into the vector requires the proper ligation that utilizes restriction sites to cut specific sites allowing the integration of topAcysB gene into an operable pTrc99A. The new plasmid enters a bacterial host through transformation in order to achieve the expression of the topA gene. Selection pressures are used to obtain the proper recombinant pTrc99A/topAcysB plasmid within a cell. Transformant cell cultures induced with IPTG, stimulate the production of topoisomerase I. Once IPTG binds to the lac repressor, it makes way for the trc promoter and topoisomerase I is produced. Topoisomerase I is extracted out the cell and compared to uninduced cells to verify the mechanisms that read and express the topA gene within pTrc99A. Lowry assay, polymerase chain reaction, absorption assays, and gel electrophoresis are implemented to view the fragments found in recombinant cells that encode topA gene and produce active topoisomerase I.
Introduction
Topoisomerase I is an enzyme that uses its unique structure to relax supercoiled DNA in prokaryotic and eukaryotic cells. Typically, topoisomerase I removes negative coils by units of one, and is often found at sites that are undergoing replication or transcription. Topoisomerase I is made up of four domains that contribute to the enzymatic activity. The active site in topoisomerase I binds to the nucleotides near the Rossmann fold in domain I, cutting the DNA strand, and relaxing the DNA. Although there are 865 amino acids that make up topoisomerase I, there are s...
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...thin pTrc99A/topAcysB plasmid, then the primers would not adhere to the exclusive sites found within topAcysB gene and the ligation of this product into M13mpI9 would not happen. To verify whether topAcysB was cloned, the multiple cloning site embedded in the lacZ ' gene of M13mp19 prevents alpha-complementation when DNA is cloned into one of the restriction sites.(1) Therefore, a blue plaque on media forms when M13mpI9/topAcysB is not ligated successfully, and white plaques will appear if M13mpI9/topAcysB was ligated successfully. The plates retrieved from this procedure exhibited white plaques, data not shown in the results, but having white plaques present, illustrates that topA gene had been cloned through PCR. The pTrc99A/topAcysB plasmid has been proven to be a useful expression vector, and has multiple applications in the process of forming topoisomerase I.
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Water was sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57ng uncut pET-41a (+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control that consisted of only sterile water. Ligation five lacks DNA ligase but has the same properties of the 1:3 molar ratio pET-41a (+)/EGFP vector.
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The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.
... starts relaxing the supercoils and altering of DNA and interacts with DNA helicase SGS1 and plays a role in DNA recombination, also cellular aging and maintenance of genome stability. Alternate splicing results in multiple transcript variants. Additional spliced variants of the gene have been described, but their complete length is unknown.
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By utilizing, and, if possible, modifying this special DNA structure, one may see a reduction of age related illness, diseases, and signs of aging. In this review of human telomeres, we will discuss the roles and functions of the telomere, its structure, and the relation of telomere length to aging and tumorigenesis. Role and Functions of The Telomeres Telomeres are special DNA structures that consist of repetitive nucleotide sequences, which serve as a “cap” to protect the ends of the chromosomes. These repetitive sequences can range from thousands of base pairs in Vertebrates to about a few hundreds of base pairs in yeast cells (Oeseburg, et al. 2009). The 'Secondary' of the 'Secondary' of the 'Secondary' of the 'Secondary' of the 'Secondary' of the 'Secondary' of the 'Secondary' of the 'Secondary' of the 'Secondary' of the 'S Located at the ends of the chromosomes, the telomeres serve as a biological life line for cells.
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