Genetic Engineering Lecture PDF

GENETIC ENGINEERING Prepared: Mary Joy P. Araneta, SST-I Learning Competency: Outline the processes involved in genetic engineering (STEM_BIO11/12-IIIa-b-6). Specific Objectives: The learner will be able to: 1. describe the processes of genetic engineering; and 2. identify the methods used...

GENETIC ENGINEERING Prepared: Mary Joy P. Araneta, SST-I Learning Competency: Outline the processes involved in genetic engineering (STEM_BIO11/12-IIIa-b-6). Specific Objectives: The learner will be able to: 1. describe the processes of genetic engineering; and 2. identify the methods used for inserting plasmids into the host cells; What is Genetic Engineering?  GENETIC ENGINEERING a gene modification process wherein the Deoxyribonucleic acid (DNA) is transferred from one organism to another. Genetic engineering, also called recombinant DNA technology, involves the group of techniques used to cut up and join together genetic material, especially DNA from different biological species, and to introduce the resulting hybrid DNA into an organism in order to form new combinations of heritable genetic material. Gene: A segment of DNA that encodes a protein and is the basic unit of heredity. Genes are the building blocks for your body. Some genes give the instructions to make proteins. A protein’s job is to tell your body what types of physical characteristics you should have, like your hair and eye color. What are chromosomes? Chromosomes are structures that look like thread, which live in the nucleus (center) of cells. Chromosomes contain DNA and protein, and they come in different sizes. Proteins called histones allow them to pack up small enough to fit in a nucleus. Without histones, our chromosomes would be as long (tall) as we are! Chromosomes give your cells the actual instructions to make you into a unique person. What is DNA? Deoxyribonucleic acid (DNA) is the material that exists in every cell in your body that holds your genetic code. It makes up your body’s instruction manual. What is DNA made of? DNA has a language that it uses to write your instruction manual (a code). Four chemical bases make up your DNA language including: Adenine (A). Cytosine (C). Thymine (T). Guanine (G). Allele A variant of a gene that controls a specific trait. Alleles are different forms of a gene that can occur at a specific location on a chromosome. For example, one allele might code for brown eyes, while another allele might code for black eyes. Homozygous Inheriting the same versions (alleles) of a gene from both parents. For example, a plant with two copies of the allele for yellow flowers is homozygous for flower color. Heterozygous Inheriting different versions of a gene from each parent. For example, a person with one allele for brown hair and one allele for red hair is heterozygous for hair color. Trait A characteristic of an organism that can be described or measured. Traits can be physical or behavioral. For example, eye color, hair color, and hairline shape are traits. Multiple alleles Occur when there are more than two different versions of a gene for a trait in a population. This can lead to a wide variety of phenotypic traits, such as blood type, eye color, and hair color. Genotype The combination of alleles that an organism carries. An organism's genotype is homozygous if the paired alleles are the same, and heterozygous if the paired alleles are different. Phenotype The physical or chemical expression of an organism's genes. Dominant Dominant version (allele) of a gene shows its specific trait even if only one parent passed the gene to the child. When a child inherits dominant brown‐hair gene form (allele) from dad, the child will have brown hair. Recessive Recessive gene shows its specific trait when both parents pass the gene to the child. When a child inherits recessive blue‐eye gene form (allele) from both mom and dad, the child will have blue eyes. In guinea pigs, black fur (B) is dominant over white fur (b), and rough fur (R) is dominant over smooth fur (r). What are the phenotypic ratios if two heterozygous guinea pigs (BbRr × BbRr) are crossed? In summer squash, white fruit color is dominant over yellow fruit color and disk-shaped fruit is dominant over sphere-shaped fruit.If a squash plant homozygous for white,heterozygous disk- shaped fruit is crossed with a plant homozygous for yellow, sphere-shaped fruit, what are the resulting genotypes and phenotypes and in what proportion? Classical Breeding focuses on the mating of organisms with desirable qualities or traits Genetic Engineering involves molecular techniques to modify the traits of a target organism. Genetically modified organisms have been subject for public scrutiny whether it is safe to use or ethically accepted. These challenge the researchers to prove the significance of GMOs as a breakthrough in science. Tools of Recombinant DNA Technology The tools mainly include the following: 1. Enzymes restriction enzymes – help to cut polymerases- help to synthesize ligases- help to bind. DNA polymerase (DNAP) (help to synthesize) is a type of enzyme that is responsible for forming new copies of DNA, in the form of nucleic acid molecules. Nucleic acids are polymers, which are large molecules made up of smaller, repeating units that are chemically connected to one another. DNA is composed of repeating units called nucleotides or nucleotide bases. DNA polymerase is responsible for the process of DNA replication, during which a double-stranded DNA molecule is copied into two identical DNA molecules. DNA ligase (help to bind) is a DNA-joining enzyme. It is a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. 2. Vectors. It help in carrying and integrating the desired gene. These form a very important part of the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the desired gene into the host organism. Most common vectors in rDNA technology Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have very high copy number. The vectors are made up of an origin of replication- This is a sequence of nucleotide from where the replication starts, a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted. 3. Host Organism. It is into which the recombinant DNA is introduced. The host is the ultimate tool of recombinant DNA technology which takes in the vector engineered with the desired DNA with the help of the enzymes. There are a number of ways in which these recombinant DNAs are inserted into the host, namely – microinjection, biolistic or gene gun, alternate cooling and heating, use of calcium ions, etc.  Methods of Introducing Plasmids into the Host Organism 1. Biolistics – the method of directly shooting DNA fragments into cells using a device called a gene gun. - This technique uses a “gene gun” to fire DNA-coated pellets on plant tissues. Cells that are able to survive and take up the expression plasmid coated pellets can acquire the ability to express the designed protein. 2. Plasmid insertion by Heat Shock Treatment - is a process used to transfer plasmid DNA into bacteria. The target cells undergo a pretreatment procedure to increase the pore sizes of their plasma membranes. The pretreatment using Calcium chloride makes the cells “competent” for the introduction of the plasmid DNA, then the cells are incubated with the desired plasmid at about 4°C for about 30 minutes. During this time, the plasmids concentrate near the cells. Afterward, a “Heat Shock” is done on the plasmid-cell solution by incubating it at 42°C for 1 minute then back to 4°C for 2 minutes. The rapid rise and drop of temperature increase and decrease the pore sizes in the membrane, respectively. The plasmid DNA near the membrane surface is taken into the cells by this process. The cells that took up the plasmids acquire new traits and are called “transformed bacterium”. 3. Electroporation - This technique follows a similar methodology as Heat Shock Treatment but uses electric shock to expand the membrane pores. This method is commonly used for the insertion of genes into mammalian cells.  Methods to Screen Recombinant Cells 1. Selection of plasmid DNA containing cells - A selection marker within the inserted plasmid DNA sequence allows the selection of “transformants”. Usually, an antibiotic resistance gene is included in the plasmid DNA. This mechanism allows only “transformed” cells to survive in the presence of the antibiotic (e.g. ampicillin). 2. Selection of transformed cells with the desired gene -Certain inserted genes within the plasmids provide visible proof of their presence. These include the antibiotic resistance genes that allow for the selection of the transformed cells within the solution. Some inserted genes also produce colored or fluorescent products that label the colonies/cells with the inserted gene. In some cases, the location of the cloning site within the plasmid is in the middle that generates a (blue) colored product in the presence of a substrate (i.e. isopropyl β-D-1 thiogalactopyranoside, or IPTG). Cells transformed with these “empty” plasmids will turn blue in the presence of IPTG. Insertion of a gene in the cloning site disrupts the sequence of the β-galactosidase gene and prevents the generation of the colored product in the presence of the substrate. Cells transformed with the disrupted β-galactosidase gene will remain “white” in the presence of IPTG. This “blue-white screening” protocol is thus able to screen for cells that were transformed with the desired gene in the cloning site.  What is recombination? Recombination is a term scientists use to define a process of forming a new combination of genes by rearranging genetic material. It can occur naturally by a) crossing over in chromosomes during meiosis, b) union of genes from male and female parents during sexual reproduction, and c) in all types of genetic exchange in prokaryotes. The method can also be done artificially by joining segments of DNA from different organisms – as a result of genetic engineering. This means that there is human intervention in the process of combining genes to achieve a desired result. This way of manipulating the molecular basis of inheritance is collectively called Recombinant DNA Technology or Biotechnology (Audesirk and Audesirk).  What is Recombinant DNA? Recombinant DNA or rDNA is a molecule of DNA that has been altered or changed, either through a natural process or through laboratory techniques. In the perspective of biotechnology, rDNA is the artificial or uncommon union of DNA fragments from two different sources of genetic material. The organism that serves as the source of the desired DNA section is called the donor, while the organism whose DNA is modified is called the vector.  The experimental manipulation of genetic materials to produce rDNA is now what called recombinant DNA technology (Schlichte). The resulting organism that carries the transgene or the artificially inserted gene is called a transgenic organism or a genetically modified organism or GMO. The new DNA molecules formed from different species by laboratory methods are of value to science, medicine, agriculture, and industry.  Applications and Products of Recombinant DNA Technology  Table 1. Applications and Products of Recombinant DNA Technology A. Medicine and Health Products What is it? a) Insulin It is a hormone made up of protein that is secreted in the pancreas by islet cells; responsible for controlling glucose (sugar) level in humans. If a person has low amount of insulin in his body, he will suffer from a disease called diabetes. Nowadays, human insulin is readily available in the market. Synthetic insulin is developed by using bacteria as vectors and host cells.  Products What is it? b) Vaccine It is a biological substance prepared from the suspension of weak or dead pathogenic (disease-causing) cells. It is introduced in the body to enhance the production of antibodies (a protein in our body that detects harmful substances) against particular antigens (any substance that may cause harm to our body) such as rabies, measles, flu, colds, herpes, Covid-19 virus and others.  Products What is it? c) Antibiotics They are chemical substances used against bacterial infections. They are produced by cultivating and manipulating fungal cells.  B. Agriculture Products What is it? Animals which are engineered to carry genes a) Transgenic from other organisms are called transgenic Animals animals. Examples are cow, sheep and goat that are modified to have human proteins in their milk. Transgenic fishes like carp, cat fish and salmon contain human growth hormones. Take a look at Figure 3 below. It shows the first genetically modified animal that has been approved for human consumption in 2015 in USA. The larger salmon is a sister to the smaller one, but the bigger fish has a gene for faster growth and although both salmon would reach the same size at maturity, the smaller fish will take longer time to reach the mature size. Genetically modified salmons will take only 18 months instead of 3 years to grow to their full-grown size, thus reducing the needed resources for raising them. Here in Caraga region, some examples of transgenic animals that are being raised by local farmers are pigs, goats and chickens. Provinces that are top hog and poultry raisers are Surigao del Norte, Agusan del Norte, Agusan del Sur and Surigao del Sur. Breeders prefer transgenic fowls because they are bigger, have high resistance to diseases and produce larger eggs. Transgenic pigs grow fast and bigger, and are low in fat. They also digest nutrients better, thereby reducing the wastes they produce.  Products What is it? Many genetically b) Transgenic Plants transgenic plants are designed to be resistant to diseases, pests, herbicides, and droughts. The Bt corn (Figure 6) was the first genetically modified crop legalized in our country in 2002. We are the first in Asia to approve commercial farming of a transgenic plant to be used as animal feed and food. The Bt eggplant (Figure 7) is the first transgenic crop created by Filipino scientists from the University of the Philippines Los Baños-Institute of Plant Breeding (UPLB-IPB). Both Bt corn and Bt eggplants are sold in local markets and are grown in many parts of our country, including Caraga. The acronym Bt stands for Bacillus thuringiensis - a common soil bacterium that contains a gene that produces a protein harmful to fruit and shoot borer insects. Scientists have incorporated this gene to corn and to eggplant to increase their resistance to pests. Another example of a transgenic plant that is common in our region is the Cavendish banana (see Figure 8). They are genetically engineered to resist infection from a fungus called Fusarium wilt tropical race 4 or TR4 that could wipe out this type of plant. Once a banana plant is infected with TR4, death is inevitable and the spread of disease from one plant to the next is fast. A gene from a worm that is resistant to fungal attacks has been inserted to the Cavendish banana’s genes; thus making the new plant resistant to TR4 infection. Caraga Region is among the top suppliers of bananas and Soriano Banana Plantation in Cabadbaran City is one of its producers.  C. Industry Products What is it? The development of important chemical compounds, improve fermentation process and proteins from waste a) Important Chemical materials can be attained by designing and developing Compounds more efficient varieties of microscopic organisms. Effective and cheap biopharmaceutical products particularly, therapeutic proteins to treat diseases in plants and animals are also great contribution of rDNA technology to industry. b) Improved Fermentation Recombinant DNA methods are also employed as answer to Process environmental issues by converting wastes into biofuels, cleaning oil spills and other toxic wastes, and by detecting arsenic and other contaminants in drinking water. c) Protein Production from Wastes