Microbial genetics lectures 22,23, and 24
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This document provides an overview of genetic engineering and its applications to microorganisms. It defines genetic engineering as the direct manipulation of an organism's genome using biotechnology. The key steps involved are isolating the gene of interest, inserting it into a vector, introducing the vector into a host cell, and harvesting the gene product from the clone. Common hosts used are bacteria, yeast, plant and animal cells. The document also discusses some tools used in genetic engineering like restriction enzymes and DNA ligase. It outlines several applications of genetic engineering in medicine, research, agriculture and industry. It concludes by noting some ethical and safety concerns regarding genetically modified organisms.
Microbial genetics lectures 22,23, and 24
- 1. Microbial genetics Bio 433 By Dr. Mona Othman Albureikan
- 2. Genetic engineering and microorganisms • Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology.
- 3. • Also called, gene splicing, gene cloning, molecular cloning, and DNA recombination. • Process cutting a gene out of a DNA strand and inserting the gene into another DNA strand. Genetic engineering and microorganisms
- 4. • It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes to produce improved or novel organisms. • The simple addition, deletion, or manipulation of a single trait in an organism to create a desired change. Genetic engineering and microorganisms
- 5. • New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. • Genes may be removed using a nuclease. Genetic engineering and microorganisms
- 6. • An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). • The first GMOs were bacteria generated in 1973 and GM mice in 1974. Genetic engineering and microorganisms
- 7. • Insulin- producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994. Genetic engineering and microorganisms
- 8. • GloFish, the first GMO designed as a pet, was first sold in the United States in December 2003. Genetic engineering and microorganisms
- 9. 1. Gene of interest (DNA) is isolated (DNA fragment). 2. A desired gene is inserted into a DNA molecule – vector (plasmid, bacteriophage or a viral genome) 3. The vector inserts the DNA into a new cell, which is grown to form a clone. (bacteria, yeast, plant or animal cell) 4. Large quantities of the gene product can be harvested from the clone. An Overview of Recombinant DNA Technologies
- 10. Tools for Genetic engineering (1. Restriction Enzymes) • Naturally produced by bacteria – restriction endonucleases • Natural function - destroy bacteriophage DNA in bacterial cells • Cannot digest host DNA with methylated C (cytosine) • A restriction enzyme • Substrate –DNA -recognizes one particular nucleotide sequence in DNA and cuts the DNA molecule (breaks down the bond between two nucleotides). sticky ends blunt ends
- 11. • DNA ligase is a enzyme that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA). • Naturally DNA ligase has applications in both DNA replication and DNA repair and Needs ATP. • DNA ligase has extensive use in molecular biology laboratories for genetic recombination experiments. Tools for Genetic engineering (2. Ligase)
- 12. Small pieces of DNA used for cloning (the gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly) Tools for Genetic engineering (3. Vectors)
- 13. 1. Self-replication - able to replicate in the host (origin of repliction). 2. Cloning site (site for recognition of restriction nucleases). 3. Promoter (and operator) - to support the gene (new DNA) expression in the host. 4. Selectable marker – antibiotic resistance 5. Proper size. Tools for Genetic engineering (Requirements of the Vector)
- 15. Hosts for DNA recombinant technology 1. Bacteria - E. coli - used because is easily grown and its genomics are well understood. • Gene product is purified from host cells.
- 16. 2. Yeasts - Saccharomyces cerevisiae • Used because it is easily grown and its genomics are known • May express eukaryotic genes easily. • Continuously secrete the gene product. • Easily collected and purified. Hosts for DNA recombinant technology
- 17. 3. Plant cells and whole plants • May express eukaryotic genes easily • Plants are easily grown - produce plants with new properties. 4. Mammalian cells • May express eukaryotic genes easily • Harder to grow • Medical use. Hosts for DNA recombinant technology
- 18. Hosts for DNA recombinant technology
- 19. 1.Transformation * Treatment make cells competent to accept foreign DNA (CaCl2 make pores in cell membrane) 2. Electroporation *Use electrical current to form microscopic pores in the membranes of cell. Insert the naked DNA into a host cell
- 20. 3. Protoplast fusion – yeast, plants and algal cells 4. Microinjection 5. Gene gun. Insert the naked DNA into a host cell
- 21. Copying the genetic material of interest - PCR • Polymerase Chain Reaction (PCR) • A reaction to make multiple copies of a piece of DNA enzymatically • Polymerase – enzyme is DNA polymerase from Thermus aquaticus – Taq polymerase. • Taq's optimum temperature for activity is 75-80°C • Can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C.
- 22. PCR reaction mixture : 1. Target DNA (template) 2. Short primers- to hybridize to the 5’ end of each DNA strand 3. four NTP – ATP, GTP, TTP, and CTP 4. Buffer 5. DNA Taq Polymerase (enzyme). Copying the genetic material of interest - PCR
- 23. Cycle program in PCR machine: 1. Denaturation - 95ºC. 2. Annealing (hybridization)- 60-65 ºC. 3. Polymerase reaction -72 ºC Copying the genetic material of interest - PCR
- 24. Applications of genetic engineering 1- Therapeutic Applications (Medicine and Industrial fermentation). • Produce human proteins – hormones and enzymes ( Insulin, human growth hormone HGH and drugs). • Vaccines ( Cells and viruses can be modified to produce a pathogen’s surface protein like Influenza, and Hepatitis B. • Gene therapy can be used to cure genetic diseases by replacing the defective or missing gene.
- 26. 2- Research • Genetic engineering is used to create animal models of human diseases. • Genetically modified mice are the most common genetically engineered animal model. • They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, and Parkinson disease. • Potential cures can be tested against these mouse models. Applications of genetic engineering
- 27. 3- Gene therapy • Gene therapy is the genetic engineering of humans, generally by replacing defective genes with effective ones. This can occur in somatic tissue or germline tissue. Applications of genetic engineering
- 28. Gene therapy
- 29. 4- Industrial • Using genetic engineering techniques one can transform microorganisms such as bacteria or yeast, or transform cells from multicellular organisms such as insects or mammals, with a gene coding for a useful protein, such as an enzyme, so that the transformed organism will overexpress the desired protein. Applications of genetic engineering
- 30. 4- Industrial • These techniques are used to produce medicines such as insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) , fuels, cleaning up oil spills, carbon and other toxic waste and detecting arsenic in drinking water. Applications of genetic engineering
- 31. 5- Agriculture One of the best-known and controversial applications of genetic engineering is the creation and use of genetically modified crops or genetically modified organisms, such as genetically modified fish, which are used to produce genetically modified food and materials with diverse uses. There are four main goals in generating genetically modified crops. Applications of genetic engineering
- 32. 5- Agriculture • To be realized commercially, is to provide protection from environmental threats, such as cold (in the case of Ice-minus bacteria), or pathogens, such as insects or viruses, and/or resistance to herbicides. • To modify the quality of produce by, for instance, increasing the nutritional value or providing more industrially useful qualities or quantities. The Amflora potato, for example. Applications of genetic engineering
- 33. • Driving the GMO to produce materials that it does not normally make. One example is "pharming", which uses crops as bioreactors to produce vaccines, drug intermediates, or drug themselves. • Another goal in generating GMOs, is to directly improve yield by accelerating growth, or making the organism more hardy (for plants, by improving salt, cold or drought tolerance). Some agriculturally important animals have been genetically modified with growth hormones to increase their size. Applications of genetic engineering
- 34. Ethical and safety • Ethical and safety concerns have been raised around the use of genetically modified food. • A major safety concern relates to the human health implications of eating genetically modified food, in particular whether toxic or allergic reactions could occur.
- 35. Ethical and safety • Gene flow into related non-transgenic crops, off target effects on beneficial organisms and the impact on biodiversity are important environmental issues. • Ethical concerns involve religious issues, corporate control of the food supply, intellectual property rights and the level of labeling needed on genetically modified products.
- 36. References Molecular Genetics of Bacteria ( 4th Edition ) (2013), Larry Snyder , Joseph E. Peters , Tina M. Henkin , Wendy Champness ISBN 10: 1555816274 ISBN 13: 9781555816278. Molecular Genetics of Bacteria, 5th Edition, by Jeremy W. Dale, Simon F. Park ,April 2010, ©2010. Genetics of Bacteria, Sheela Srivastava,(2013) ISBN: 978-81-322-1089-4 Microbial Genetics. (1994). Jones and Bartlett Series in Biology. Jones and Bartlett Publishers, Inc.; 2nd edition, ISBN-10: 0867202483, ISBN-13: 978-0867202489, 484 pages. Microbial genetics. (2008). Jones and Bartlett series in biology Series of books in biology. David Freifelder, publisher, Jones and Bartlett, 1987. 601 pages. Molecular Biology: Genes to Proteins Hardcover . (2007). Burton E. Tropp, Publisher: Jones & Bartlett Publishers; 3 edition, ISBN-10: 0763709166, ISBN-13: 978-0763709167, 1000 pages .