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Home work 3 (Lab) Microbial Genetic
1. Describe the usefulness of gene cloning also give an example of your answer - Cloning is the producing many identical copies of the same recombinant must be replicated many times to provide material for analysis, sequencing, etc. The term 'cloning' have entered popular vocabulary of many people. The issues on cloning are getting controversial and growing more complicated the word "clone" comes from the Greek "klwn" which means similar. For example cloning usefulness NEW YORK — It may be futile to try producing stem cells by putting human DNA into cow or rabbit eggs and making hybrid cloned embryos, a strategy that triggered controversy recently in Britain. Cloning gives parents the opportunity to choose what characteristics they want their children to have. For instance, the parents want their child to have Albert Einstein’s IQ. The extraordinary athleticism of Michael Jordan. Horticultural: The term clone is used in horticulture to refer to descendants of a single plant which were produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. The hormone insulin or growth hormones, the first hormone that is produced by this method is that insulin can be synthesized in bacteria E. coli.
3. Apart form bacterial host is there any other host cell that we can use? If so, what are they? (provide the paper conclude abstract page) to support your answer Definition noun, plural: host cells -A cell that harbors foreign molecules, viruses, or microorganisms. For example, a cell being host to a virus. - A cell that has been introduced with DNA (or RNA), such as a bacterial cell acting as a host cell for the DNA isolated from a bacteriophage. Other host cell (Cell Culture), (Embryonated egg) and (Experimental animal)
Miss Phratchaya Seeladlao. ID: 5231105037 Iv model in eukaryotic cell used yeast, Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process.
Understanding the yeast host cell response to recombinant membrane protein production.
Bawa Z, Bland CE, Bonander N, Bora N, Cartwright SP, Clare M, Conner MT, Darby RA, Dilworth MV, Holmes WJ, Jamshad M, Routledge SJ, Gross SR, Bill RM.
Source School of Life and Health Sciences, Aston University, Birmingham, U.K.
Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.
Miss Phratchaya Seeladlao. ID: 5231105037
5. Compare a contrast the protocols used the extract DNA, RNA and protein - The identification and validation of molecular markers DNA, RNA and micro RNA based on biomarkers and when compared the DNA/RNA obtained with the optimized protocol and they include variable cell lysis times and different chemistries for the extraction of RNA and DNA in addition, deparaffinization in the RNA/DNA extraction protocol by Hurt et al. Furthermore, to process many samples in parallel, a miniaturization of the protocol was required. However, when we applied the protocol with the modifications, the RNA obtained was frequently degraded. Since RNA is often preserved as ethanol precipitate, we explored the idea of adding RNA protecting substances such as ethanol, isopropanol or the denaturing solution used by Hurt et al. already before breaking up the cells by means of a bead beating step. Agarose gel electrophoresis are differ the concentration base on size of biomolecule (DNA/RNA extraction from FFPE tissue samples), - In Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution, extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type (A comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins).
6. Compare & contrast the protocols used to extract genomic DNA from bacteria, fungi, plant and animal cells The goal of genomic DNA isolation depends on what the applications of the DNA after isolation. Purity, source, quantity and quality of DNA are all issues that need to be addressed prior to genomic DNA extraction. A whole host of different methods, technologies and kits are available now to researchers to isolate genomic DNA from cells. - Quantity of DNA needed - Molecular weight and size of DNA - Purity of DNA required - Time available - Expense or money available - Downstream applications of DNA - Ease of DNA extraction technique or method
Miss Phratchaya Seeladlao. ID: 5231105037 Several DNA extraction method are widely used to isolate DNA from bacteria and yeast including phenol extraction but they often involve multiple, time consuming steps including the handling of toxic chemicals (Ausbel et al., 1995). DNAzol is a complete and ready to use reagent for the isolation of genomic DNA from solid and liquid samples of animal and plant origin. This reagent is an advanced DNA isolation method (Chomczynski et al., 1997) that combines both reliability and efficiency with simplicity of the isolation protocol. The DNAzol procedure is based on the use of a novel guanidine-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from a cell lysate. The protocol is fast and permits isolation of genomic DNA from a large number of samples of small or large volumes. Originally, the DNAzol procedure was proposed for DNA extraction from animal and plant tissues. In the present study we provide a modify DNAzol reagent protocol for the isolation and purification of total DNA from bacterial and yeast (Microbial DNAzol). Many procedures in molecular biology require the isolation of high quality genomic DNA. This study investigated a new method to extract DNA from Gramnegative, Gram-positive bacteria, Mycobacteria and yeasts. Guanidine thioisocyanate present in DNAzol is capable of binding DNA to silica particle column. Subsequently the silica with adsorbed DNA is washed to remove impurities and the clean DNA eluted in appropriate buffer. Results indicated that the new extraction method is simple and reproducible. This isolation technique is faster and easier to perform than the other conventional extraction methods. Finally the recovered DNA is of high quality and suitable for downstream applications.
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