Question 1 B) What are opportunistic pathogens and how do they differ from primary pathogens?
Answer: opportunistic pathogens are a category of microorganisms that produce infections in clinics, immunosuppressed individuals, or latent disorders such as cystic fibrosis that promote infection, not usually infecting safe hosts.Primary pathogens can cause disease-related patho-logical changes in a healthy human while opportunistic pathogens can cause disease only when they are affected by a breakdown of protective barriers or immunosuppression.In cer-tain situations, opportunistic pathogens activate primary disease pathogens and opportunistic pathogens are specific because primary pathogens may activate disease-related pathologies in a healthy person, while opportunistic pathogens may only induce illness if the individual be-comes weakened by a weakening in defensive defences or immunosuppression.
Question 1 C) List the general safety rules and regulations for working with micro-organisms in a biomedical laboratory. Explain how these rules keep you safe.
Answer: The best way of dealing around live microorganisms is by following the following guidelines 1. Each laboratory microorganism is dangerous. 2. Each fluid culture contains pathogens. 3. The harmful compounds in each fluid community are identified. 4. Wear a lab coat covering your neck, gloves and safety glasses. 5. Ensure sure the hair from the nose has been pulled back. 6. Don't eat , drink, smoke or put anything (even stylos and pencils) in your mouth. 7. Chew no gum in the classroom. 8. Should not use the water from a tap to brush sticky stickers.9. Don't touch your eyes or face. 10. Maintain the bench clean of things that are not important. 11. Drop your bags into the storage area. 12. Do not remove any of the la-boratory materials. 13. Be cautious of creating aerosols (airborne substances). 14. You must know where to discard contaminated items. Ask your teacher, if you're uncertain. 15. The sharps used have to be stored in clean containers. 16. Holding the lids as far as possible on tubes that hold cultures. Be careful not to make aerosols (for example pipetting can create aerosols too vigorously). 17. Promptly notify your tutor / ECU laboratory manager of any accidents. This helps alleviate seed fluid wounds, abrasions, or leaks. 18. Remove bench coat after class and swab the work bench equipped with 70 percent ethanol or similar. 19. Thor-oughly wash your hands before you leave the laboratory. Dry your hands with towels made from cotton. 20. Lab coats should be periodically dried, and hanging to dry in the light.
The guidelines mentioned above are very important and it is necessary that you stick rigidly to a code of conduct in microbiology to eliminate the risk of an infection that has been ob-tained by the laboratory.
Question 2 A) Describe the structure of viruses and how this is used to classify them
Answer: Structure: Nucleic acid and protein coat are the two principal components of the simplest virion.During an infection, the capsid attaches the virion onto the prospective host cell to suitable receptors.To order to shield the viral genome from the nucleus, it often serves as a cover.This is so small that a light microscope has to be used to display. Virus does not grow through binary fission or through any other form of cell division. It dose does not have organelles like ribosomes. It dose not even have some method for producing ATP. Virus may be of different shapes, including Helical, Icosahedral, and complex.
Classification of viruses is the method by which viruses are identified and put into a taxo-nomic scheme.The classification of viruses is the subject of ongoing discussion and pro-posals, similar to the classification systems used for cellular organisms.This is primarily due to the faux-living nature of viruses, i.e. particles that are not alive with certain chemicals that are similar to life or non-cell life properties.As such, they do not fit in well with the current system of cellular organisms' biological classification.
Viruses are categorized by traits that are phenotypic, like appearance, form of nucleic acid, mode of replication, host species and the variety of disease it causes. International Committee on Virus Taxonomy (ICTV) system is responsible for the systematic taxonomic classification of viruses, while the classification of Baltimore scheme could be utilized to position viruses among seven classes depending on their nature of synthesis of mRNA.The ICTV defines par-ticular designation conventions and additional classification guidelines.
Question 2 C) What is the most effective way to prevent viral infections in human popu-lations?
Question 3 A) What are primary cell lines and how can they be used?
Answer:Cells extracted by enzymatic or mechanical processes directly from human or animal tissue.Once isolated, plastic or glass containers are inserted into a specialized medium that contains essential nutrients and supports growth factors.Cell lines are typically chosen for ease since they are simple to manage and circulated.They also are less preferred as an im-portant clinical alternative, as they retain the essential character of their original tissue.
It is known that dynamic activity induces genotypical and phenotypical cell-line heterogenei-ty.
Question 3 B) How would you process a piece of organ tissue in order to develop a pri-mary cell culture?
Answer: Primary culture is the tissue-directed culture cell culture method developed.The dis-section of the biopsy of the tissue or organ begins with a primary culture(~1 cm3).Cells in-clude tissue organization connections between cellular and intercellular basal membranes or matrix.First of all, such connections (single cell suspension) need to be eliminated for cultiva-tion of cells.The division of cells includes the usage of multiple proteolytic enzymes such as trypsin or collagenase and techniques of mechanical separation such as the splitting of tissue using surgical knives.The cell suspensions obtained are therefore purified and transferred to cultural vessels by serial dilution and centrifugation processes.Many cells are then released from cellular links and stick to a culture vessel.The process of primary culture is completed if non-sticky cells and their residuals are separated from the cell environment.In addition, with the exception of the cells originally intended to be obtained in primary cultures, the vessel of culture is often present in specific cell types , particularly fibroblasts.
Question 3 C) What is Hayflick’s phenomenon?
Answer: The Hayflick restriction or Hayflick impact is the amount of cycles before the cell division is halted a normal group of human cells.American anatomist Leonard Hayflick ad-vanced the concept of the Hayflick limit at the Wistar Institute in Philadelphia, Pennsylvania , United States, in 1961.The theory of Hayflick helps describe the mechanism behind the ag-ing of cells. The principle suggests that a typical human cell can multiply and differentiate just forty to sixty times until it can no longer split and break down through programs causing cell death or apoptosis.Then, in secondarily obtained cultures, it is possible to assure that a certain type of cell proliferates and pushes others based on growth factors unique to the de-sired cell type.Cells are very important in primary culture as they represent the closest cell forms in normal tissues.However, sometimes these disadvantages may be combined.Of these, the most important is a limited lifespan.These cells can be sequentially replicated and re-tained.
Question 3 D) What are the main differences between stem cells and differentiated cells. In your answer mention THREE (3) types of stem cells.
Answer:Two different cell classes involved in cellular development are steam cells and sepa-rate cells.Stem cells are regenerated continuously throughout the lifetime of the organism.The major differentiation between stem cells and differentiated cells is that stem cells are the non-specific cells able to self-renew and transform into mature cells, while differentiated cells are socialized in order to play a specific role in the body.
During different stages of organism development three types of cells can be identified: em-bryonic stem cells, fatal stem cells, and adult stem cells. Stem cells, originating from the ear-ly stages of the foetus, are called embryonic cells (ESCs). Multi-potent stem cells make up the inner cell bulk, resulting in three germ layers: ectoderm, mesoderm, and endoderma.
These multi-powerful stem cells can be removed from the blastocyte in laboratory to preserve their non-specialized phases as cell culture lines.These cell lines are used in medicinal treat-ments.Fetal stem cells, which ultimately become distinct, untimed organs such as the neural tube, intestines , liver, hormone secreting glands, teeth, faces or the brain, and coordination tissues, are called primary cells of the embryo.The fetal liver and blood include hematopoiet-ic stem cells from the placenta and umbilical cord.
If blood cells, they give rise to several forms. Adult stem cells are known as undifferentiated cells found in differentiated tissue like the bone marrow and brain in adults.Human stem cells regenerate themselves by the development of identical copies over the lifespan of humans.In addition, the tissue is divided into specialized cells.In addition to the bone marrow and spine, blood, liver, skin, dental pulp, leg, muscle skeletal, pancreas, and the gastrointestinal tract are all contained in adult stem cells in the body.
Question 4 A) Describe the process of cutting DNA into fragments and explain how you could use electrophoresis to separate these fragments. What type of gel would you use and how would you visualise the DNA? Describe the process in detail.
Answer: A method commonly used in laboratories for separating charged molecules such as DNA, RNA , and proteins by size is Gel electrophoresis.Users can distinguish various lengths of DNA fragments with Electrophoresis.It would use agarose gels to image DNA fragments. The sum of agarose in the fluid relies on the scale of the pieces of the DNA that are function-ing.The power flow is then switched on to the positive side of the gel by the negatively modi-fied DNA.By tracking the buffer filling dye migration the extent that the DNA has migrated into the gel can be visually determined.The electric current is enough long to insure that the DNA fragments travel through the gel to isolate them, but not so long that the gel stops.The gel is made of a fluorescent colouring which binds to the DNA and is put on a trans ultravio-let light that displays the stained DNA as bright bands to visually represent the DNA.The DNA banding pattern will be noticeable if the gel is running correctly.
Question 4 B) What is the Polymerase Chain Reaction and why is it used in research and medical diagnosis? How is electrophoresis used as part of this process?
Answer: Polymerase chain reaction is a common laboratory technique used to make many copies of a DNA region. PCR depends on Taq polymerase, a thermostable DNA polymerase that includes DNA primes uniquely tailored for the DNA area of interest. In the PCR, the re-action is cycled repeatedly through a series of temperature changes which allow the produc-tion of many copies of the target region. Researcher used to test their experiments using the PCR, because it could be a gene whose function a researcher wants to understand, as a genet-ic marker used by forensic scientist to match DNA with suspects in the crime scene. It allows scientists to take a very small sample of DNA and to amplify it to a sufficiently large amount for a detailed study. This is used to amplify genes associated with patient DNA genetic de-fects, which is the justification for using the PCR in medical care, genetics science, and even some ecological branches. In addition, electrophoresis is part of this process as PCR reaction is typically visualized using gel electrophoresis, a technique in which DNA fragments are dragged by an electrical current through a gel matrix and divide DNA fragments by thick-ness.
Describe the steps in an experiment in which a gene coding for insu-lin production is inserted into a bacterial plasmid by answering the following questions
5a) How would you cut out the pieces of DNA required? What do you need to know to be able to do this?
Restriction enzymes are added to break DNA into smaller pieces, or restriction of endonucle-ase.Specific sequences of nucleotides are always cut.Similar sequences of DNA are identified and distinguished by different enzymes of restriction.Bacterias use limitant enzymes to kill viruses–the enzymes are targeted at and separated into useless pieces of viral DNA.As other enzymes, a limited enzyme acts in a shape that suits.When a DNA sequence falls into touch with a configuration that fits a portion of the enzyme, the marker, it sprays across the DNA and takes the two DNA molecule strands into breakup.Bacteria possess enzymes that are re-strictive.In the removal of viruses, bacteria utilize limiting proteins, and the viral DNA is separated into unserviceable parts.DNA consists of two nucleotide complementary strands that spiral around each other in a double helix.Enzymes that limit and divide the DNA to fragments are divided from all nucleotide chains; however, they are not both similar-ly.Restriction enzymes are DNA-cutting enzymes present in (and obtained for use from) bac-teria. Since they cut inside the molecule, they are often called endonuclease for restriction. To be able to sequence DNA, the cutting into smaller fragments is first necessary.
5b) What two laboratory techniques can be used to force a plasmid to enter inside a bacterial cell?
Answer: Two laboratory techniques can be used to force a plasmid to enter inside a bacterial cell include
1. Bacterial transformation- heat shock of chemically prepared competent cells (chemi-cal transformation) -
Chemically competent cells are mixed with plasmid DNA with chemical transformation and are briefly exposed to elevated temperature, a process known as heat shock. Firstly, cells are incubated in a polypropylene tube with DNA on ice for 5–30 minutes. Polystyrene tubes should be avoided, since DNA can adhere to the surface, thus reducing the efficiency of transformation. For best results 17 x 100 mm round-bottom tubes have traditionally been used. Using 1.5 mL microcentrifuge tubes can contribute to weak heat distribution due to nar-rower cell suspension surface-to - volume ratios, which can reduce the output of transfor-mation by as much as 60–90 percent, particularly for higher-efficient cells.
2. electroporation of electro competent cells-
Electroporation involves exposing competent cells and DNA to a brief pulse of a high-voltage electric field using an electroporator. This treatment is thought to induce transient pores in cell membranes which allow DNA to enter the cells. In bacterial transformation, the most popular type of electrical pulse is exponential decay, where a fixed voltage is added and decay over a few milliseconds, called the time constant. The voltage applied is measured by field intensity (V / cm), where V is the original peak voltage, and cm is the estimate of the distance between the used cuvette electrodes. Usually, bacterial electroporation utilizes 0.1 cm of cuvettes (volume 20–80 μL) and needs a field power of > 15 kV / cm.
5c) What extra genes can be cloned into the plasmid to help you determine which bacte-ria contain the plasmid and differentiate them from those that do not contain the plas-mid?
Bacteria that has taken up the plasmid is identified as the ones that have not will have neither the ampicillin resistance nor be bioluminescent because the Lux operon is a part of the pVIB plasmid. The -plasmid will survive only in the absence of ampicillin. The +plasmid will survive around ampicillin and in the absence of it. So the bacteria that are biolumines-cent and are not killed by the Ampicillin are the ones that have incorporated the plasmid in-troduced.
Bacteria will be transformed in the presence of an antibiotic and include the antibiotic-resitant gene along with other DNA the bacteria will retain. If the bacteria does not survive, it did not take up the plasmid
5d) Describe two applications for recombinant DNA technology. What were the aims of these projects and how were they carried out?
The three important applications are: (1) Applications in Crop Improvement (2) Applications in Medicines and (3) Industrial Applications.
1. Applications in Crop Improvement
• Distant Hybridization: Through the advent in genetic manipulation, genes from distantly related organisms may now be converted. The obstacles to gene transmission have been solved between organisms, or even genera. The desirable genes can be transferred via re-combinant DNA technology, even from lower organisms to higher organisms.
• Development of Transgenic Plants: Genetically transformed plants are called transgenic plants which contain foreign genes. Resistance to viruses, insects and rodents, herbicides, drought; resistance to metal toxicity; introduction to male sterility for plant breeding pur-poses; and quality control may be accomplished by this recombinant DNA methodology. A glaring example is BT-cotton which is resistant to bollworms.
• Development of Root Nodules in Cereal Crops: Leguminous plants have root nodules comprising the bacteria Rhizobium, which release nitrogen. This bacteria transforms the root nodules of the free ambient nitrogen into nitrates. Via genetic modification techniques, the bacterial genes responsible for this nitrogen fixation may now be passed to cereal crops such as wheat, rice , corn, barley etc. thereby rendering these crops more capable of fixing atmospheric nitrogen.
• C4 Plant Development: Increasing yields can be accomplished by improving crop plant photosynthetic performance. The photosynthetic rate can be increased by converting C3 plants into C4 plants, which can be achieved either through protoplasm fusion or recombi-nant C4 plants using DNA technology have higher biomass production potential than plants with C3. Many C4 plants are cultivated in tropical and subtropical areas (sorghum, sugar-cane, maize, some grasses).
Biotechnology, particularly genetic engineering, plays an important role in the production of medicinal antibiotics , hormones, vaccines, and interferon.
• Antibiotics Productions: Penicillium and Streptomyces fungi are used to mass-produce popular penicillin and streptomycin antibiotics. To greatly increase the yield of these anti-biotics, genetically efficient strains of these fungi were produced.
• Hormone Insulin Producing: Insulin, a hormone that is used by diabetics, is usually ex-tracted from cow and pig pancreas. This insulin slightly differs from human insulin in structure. As a result, in about 5 percent of patients it results in allergic reactions. Human insulin-producing gene has been incorporated into bacterial DNA, and such genetically en-gineered bacteria are used to produce insulin on a large scale.
• Vaccine production: Vaccines are now produced through the transfer of antigen-coding genes to bacteria-causing diseases. Such antibodies provide the same bacteria or virus to protect against the infection.
• Interferon production: Interferon's are proteins induced by viruses produced by cells in-fected with the virus. Interferon is antiviral in action and acts as the first line of defense against viruses that cause serious infections including breast cancer and malignant lymph nodes. Natural interferon is derived from the human blood cells in very low quantity. It is thus also very expensive. Interferon can now be produced at much cheaper rate by recom-binant DNA technology.
• Enzyme production
• Gene Therapy: Also, recombinant DNA technique can produce some useful enzymes. For example, the genetically engineered microorganisms have produced enzyme urikinase, which is used to dissolve blood clots. Genetic engineering may one day enable medical sci-entists to substitute normal genes for the defective genes responsible for hereditary diseases ( e.g., haemophilia, phenylketonuria, alkaptonuria). The modern treatment method is called gene therapy.
• Dispute Parenting Solution: Publicizeed cases of parentage can now be most accurately resolved by recombinant technology than by blood testing.
• Injuries and illnesses diagnosis: Recombinant DNA technology has provided a wide range of tools to help doctors diagnose illnesses. Many of these include probe construction: small single-stranded DNA fragments connected to a radioactive or fluorescent tag. For starters, food poisoning Salmonella, pus developing Staphylococcus, hepatitis virus, HIV, etc., these tests are also used to classify infectious agents. By testing the DNA of prospec-tive parents carrying genetic disorder, it is possible to determine their genotype and predict their chances of producing an afflicted child.
3. Industrial Applications: Recombinant DNA technique in industries will help in the pro-duction of commercially important chemical compounds, improving existing fermentation processes and producing proteins from waste. It can be done through the production of more productive micro-organism strains. Specially developed microorganisms can even be used to purify the pollutants. Biotechnology has many useful applications in crop improvement, med-icines and industry , especially recombinant DNA technology.
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