Genetic Engineering Notes PDF

WEEK 2: Genetic Engineering MELC: Outline the processes involved in genetic engineering. (STEM_BIO11/12-IIIa-b-6). General Biology 2 1/2 Science, Technology, Engineering, and Mathematics  Learning Objectives At the end of the lesson, you...

WEEK 2: Genetic Engineering MELC: Outline the processes involved in genetic engineering. (STEM_BIO11/12-IIIa-b-6). General Biology 2 1/2 Science, Technology, Engineering, and Mathematics  Learning Objectives At the end of the lesson, you should be able to do the following: Define genetic engineering. Enumerate the general principles of recombinant DNA technology. Explain the history, techniques, and processes involved in recombinant DNA technology. Cite applications of recombinant DNA technology. A1: Design a Pokémon Question: If you would design a Pokémon, what would it be and what are the possible unique traits it possess?  Types of Breeding Methods Classical and modern breeding techniques are all founded on the fact that the DNA molecule stores and controls the expression of the genetic information. A2: Concept of Genetic Engineering How would you define Genetic Engineering using the diagram below?  Genetic Engineering hypothetical source of hypothetical recipient of new offspring? desirable trait desirable trait There are cases wherein an organism possessing a desirable trait is reproductively incompatible to an organism needing that trait.  Genetic Engineering involves the direct Add Genes Overexpress manipulation of Genes genes of organisms in laboratory for Underexpress Genes them to express the desired traits or manifest a wide Delete Genes array of traits.  Genetic Engineering Recombinant DNA Add Genes technology is the Overexpress Genes primary technique used in the genetic Underexpress engineering resulting Genes to the creation of transgenic organisms and Delete Genes GMOs. Genetic Engineering Genetic Engineering The Macedonian Academy of Sciences and Arts has devoted their resources to establish a genetic engineering facility.  Brief History of Genetic Engineering 1972: Cohen and Boyer 1974: Rudolf Jaenisch 1976: Genentech Stanley Cohen and Herbert Boyer 1983: Bevan, Flavell, and Dell- introduced a plasmid segment into the Chilton plasmid of another bacterium.  Brief History of Genetic Engineering 1972: Cohen and Boyer 1974: Rudolf Jaenisch With their attempt to perform recombinant 1976: Genentech DNA technology, Escherichia coli became the first transgenic bacteria. 1983: Bevan, Flavell, and Dell- Other attempts were made such as inserting a Chilton fragment of frog DNA into a bacterial plasmid.  Brief History of Genetic Engineering 1972: Cohen and Boyer The recombinant DNA was initially 1974: Rudolf Jaenisch called chimera, which is named after the mythological creature Chimera — one that has a head 1976: Genentech Of a lion, the body of a goat, and tail of a snake. 1983: Bevan, Flavell, and Dell- Chilton  Brief History of Genetic Engineering 1972: Cohen and Boyer 1974: Rudolf Jaenisch 1976: Genentech He successfully created the first transgenic animal — a mouse. However, the initial problem was that the 1983: Bevan, Flavell, and Dell- Chilton recombinant DNA could not be inherited by the offspring.  Brief History of Genetic Engineering 1972: Cohen and Boyer 1974: Rudolf Jaenisch The company Genentech was able to 1976: Genentech produce somatostatin, a human protein produced by GM E. coli. 1983: Bevan, Flavell, and Dell- This protein regulate human hormones Chilton in the body.  Brief History of Genetic Engineering 1972: Cohen and Boyer 1974: Rudolf Jaenisch 1976: Genentech Two years later, insulin can already be produced by the same bacterial species 1983: Bevan, Flavell, and Dell- Chilton (GM E.Coli.)  Brief History of Genetic Engineering 1972: Cohen and Boyer 1974: Rudolf Jaenisch 1976: Genentech Richard Flavell (left) and Mary Dell-Chilton (right), alongside Michael Bevan, successfully 1983: Bevan, Flavell, and Dell- created the first transgenic crop, a tobacco Chilton making it resistant to herbicide. Principles of rDNA Technology DNA as the Proteins as blueprint of phenotypic life determiners rDNA Introduction Technology Identification of foreign of a gene of genes interest Identification of a host organism  Sample Case: The BT Corn Normal Corn Corn is a major crop in agricultural countries. Bt Corn  Sample Case: The BT Corn Corn is infested by corn borers of genus Ostrinia. Normal Corn Bt Corn  Sample Case: The BT Corn Normal Corn Infestations lead to major crop damage. Bt Corn  Sample Case: The BT Corn Normal Corn A specific gene from Bacillus thuringiensis is introduced to Through rDNA corn. technology... Bt Corn This makes BT corn resistant against corn borers.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST The Bt gene or cry1Ab gene from B. thuringiensis produces the cry protein, which can cut holes into the gut of the corn borers.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST It involves a chemical reaction that uses the restriction Endonucleases. This is a restriction enzyme EcoRI of E. coli, which can recognize and cut the sequences 3’CTTAAG5’ and 5’GAATTC3’ in the DNA of B. thuringiensis  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST Gel electrophoresis will then be performed to separate the DNA molecules according to their length. In the case of the cry1Ab gene, it is approximately 3,500 base pairs long.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST Then, a probe is used to locate the gene of interest. A probe is either a DNA or RNA sequence that is complementary to the gene of interest. Usually, a radioactive chemical tag is added to the probe to easily locate the DNA segment of interest.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST Microprojectile Electroporation bombardment Methods of Introducing Gene of Interest Agrobacterium tumefaciens- mediated transformation  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST Electroporation performed by subjecting Methods of Introducing the host cell into short Gene of electrical bursts to allow Interest the entry of DNA into the cells  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST Microprojectile bombardment Methods of Introducing In this method, gold or Gene of Interest tungsten particles are shot into the cell at high velocity. These particles are coated with DNA containing the gene of interest.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST The process of microprojectile bombardment carries out the high-velocity introduction of DNA- coated particles through pressurized helium.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST The most utilized In the case of Bt corn method of introducing Methods of and most dicots, this is the gene of interest is Introducing facilitated by the soil through a Gene of bacterial species Interest Agrobacterium bacterial cell that can infect the host cell. tumefaciens. Agrobacterium tumefaciens- mediated transformation  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST There are several features of Agrobacterium tumefaciens which make it efficient in incorporating the gene of interest into the plant host cell.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST 1. It infects plants and causes the crown gall disease, a type of tumor development. The process of infection is mediated by the introduction of the plasmid into the plant.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST 2. Ti plasmid (tumor- inducing plasmid) during infection is used as a vector or a vehicle for incorporation in the host of DNA.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST 3. During crown gall infection, the section of the Ti plasmid incorporated into the plant chromosome is called the T-DNA.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST T-DNA in the plant will be cut by endonucleases and will be isolated in a laboratory. By using DNA ligase, the cry1Ab gene is inserted into the cut region.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST In the laboratory, the recombinant plasmid will be reintroduced to the A. tumefaciens and are used to infect cultured corn cells thus incorporating the cry1Ab gene into the corn genome.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST The genetically engineered corn cells are allowed to regenerate into a mature plant, which can already be formally called Bt corn. The plant can already produce Bt toxin, which will make it deter corn borers.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST Only certain groups of insects have the receptor to this toxin; thus, BT corn is harmless to humans and other higher animals.  Process of Genetic Engineering IDENTIFICATION OF GENE OF ISOLATION OF GENE OF INTRODUCTION OF INTEREST INTEREST GENE OF INTEREST BT corn cross to other varieties of corn with a significantly high yield.  Other Examples of Genetic Engineering Animal: Transgenic Mice Mice are highly tested subjects for genetic studies. The picture shows an introduction of the gene for the green fluorescent protein (GFP) from a sea jelly.  Other Examples of Genetic Engineering Bacterial: Transgenic E. coli The advent of rDNA technology allowed E. coli to produce insulin, clotting factor VIII protein (for hemophilic), and hGH (human Growth Hormone). Insulin was the first product of rDNA technology to be made available commercially.  Other Examples of Genetic Engineering Bacterial: Transgenic E. coli Prior to rDNA technology, the only alternative sources of functional are human cadavers. To allow E. coli to produce hGH: 1. A plasmid from the E. coli is used as a vector. 2. Restriction enzymes are also used to cut the plasmid into its appropriate region, and DNA ligase is used to insert the hGH into the plasmid. 3. The recombinant plasmid will then be reintroduced in the bacterial cell. Genetically engineered E. coli can be cultured for them to transmit a copy of the hGH gene to succeeding cell generations. They are then stimulated to synthesize hGH, which is eventually purified for commercial purposes. Remember In the application of recombinant DNA technology to produce transgenic crops, always remember the essential steps to incorporate the foreign gene into the host cell as follows: (1) determination of the gene of interest, (2) isolation of the gene of interest by using restriction enzymes and gel electrophoresis, Remember In the application of recombinant DNA technology to produce transgenic crops, always remember the essential steps to incorporate the foreign gene into the host cell as follows: (3) use of probe to locate the gene, (4) use of restriction enzymes and DNA ligase to combine the gene and plasmid, Remember In the application of recombinant DNA technology to produce transgenic crops, always remember the essential steps to incorporate the foreign gene into the host cell as follows: (5) reintroducing the recombinant DNA into a bacterial cell, (6) allowing the bacteria to transform the host plant cells, and (7) regenerating the cultured plant cells. Let’s Sum It Up! The recombinant DNA technology is a continuous effort to improve methods in identification, isolation, and introduction of the gene of interest. Novel applications are also being identified to address concerns in agriculture, pharmaceuticals, and medicine.  Quipper Exercise: DIRECTIONS: Determine the accuracy of each of the following statements. Write true if the statement is correct and false if otherwise. 1. Genetic engineering involves the manipulation of the genome of an organism for it to manifest or express the desired trait. 2. Recombinant DNA technology involves the manipulation of genes either by over-expressing them or under-expressing them. 3. Werner Arber and his colleagues were among the first to successfully create a transgenic organism. 4. The first successfully created genetically modified organism is a bacterium. 5. Bacterial cells were successfully genetically manipulated to produce human proteins such as human growth hormone and insulin.  Quipper Exercise: DIRECTIONS: Determine the accuracy of each of the following statements. Write true if the statement is correct and false if otherwise. 6. BT corn is created using a gene from Bacillus thuringiensis to obtain resistance against locusts. 7. Restriction endonucleases are important raw materials in genetic engineering because they allow the cutting of DNA molecules, which is especially important in obtaining the gene of interest. 8. Plasmid serves as the vector of the gene of interest for the latter to be incorporated into the host genome. 9. The tumor-inducing capacity of the plasmid of the soil bacterium Agrobacterium tumefaciens is utilized in the genetic engineering of crops. 10. Incorporating the foreign gene at a stage later than the zygote will make a mouse fully transgenic.  Seatwork #2 I. DIRECTIONS: 1. What is genetic engineering? 2. What is the difference between transgenic organism and genetically modified organism? 3. Research for an example of either transgenic organism or genetically modified organism. Draw it in your notebook, explain its unique traits and how humans benefited from this.  Seatwork #2 II. DIRECTIONS: Arrange the following steps in the genetic engineering of corn to acquire pest resistance. Write letters from A to J to indicate the correct sequence. Strictly no viewing from the PPT. (NOTEBOOK) 1. Allow the transformed Agrobacterium tumefaciens cells to infect the cultured corn cells. 2. Cross the mature Bt corn with other superior corn varieties. 3. Obtain and culture Bacillus thuringiensis cells as sources of the cry1Ab gene. 4. Determine the source of the gene of interest which expresses the pest resistance trait. 5. Isolate the genetic material from Bacillus thuringiensis. 6. The tumor-inducing plasmid will be incorporated into the genome of the infected corn cells. 7. Insert the cry1Ab gene into the tumor-inducing plasmid of Agrobacterium tumefaciens by using ligase. 8. Treat the isolated genetic material with restriction enzymes and subject it to gel electrophoresis and probes to identify and isolate the cry1Ab gene. 9. Regenerate the cultured corn cells and allow them to grow into mature Bt corn. 10. Reintroduce the recombinant plasmid into Agrobacterium tumefaciens cells.