ABT11 Lecture 2 - Principles of Modern Biotechnology PDF

Copyright EM Tecson-Mendoza 2010-2018 Principles of Modern Biotechnology Carlo Miguel C. Sandoval, PhD Roberta N. Garcia, PhD Eureka Teresa M. Ocampo, PhD Evelyn Mae Tecson-Mendoza, PhD Learning objectives To discuss the basis and principles of genetic engineering...

Copyright EM Tecson-Mendoza 2010-2018 Principles of Modern Biotechnology Carlo Miguel C. Sandoval, PhD Roberta N. Garcia, PhD Eureka Teresa M. Ocampo, PhD Evelyn Mae Tecson-Mendoza, PhD Learning objectives To discuss the basis and principles of genetic engineering To understand the steps involved in genetic engineering To learn about current and future products of modern biotechnology  Genetic engineering of animals Genetic engineering of plants Modern Recombinant medicines “DNA simple2” by Forluvoft. Creative Commons CC0 license Recombinant microorganisms Recombinant vaccines Recombinant diagnostics, DNA markers GMO(2) by Prince TC. Creative Commmons Protein markers Attribution-Share Alike 4.0 International License Cell culture Immunodiagnostics Plant tissue culture Classical “Plant tissue cultures, National Center for Genetic Resources Preservation, USDA” by Lance Cheung. Creative Commons CC0 License Vaccines Antibiotics Biofertilizers, biological nitrogen fixation, fermentation “Wine and Cheese 6995 (2008)” by Narek75. Creative Commmons Attribution-Share Alike 4.0 International License ©EMTMendoza 2019 Ancient Classical Modern I. Basis of Genetic Engineering What is a gene? What are the information encoded in genes converted into traits? What is recombinant DNA technology or genetic engineering genome editing? In 1866, Gregor Mendel discovered important concepts of genetics Traits are inherited from one generation to another Elementen (genes) control the traits of an organism “Gregor Mendel” by Wilfredor. Creative Commons CC0 License There is recombination of parental traits in the offspring Some genes are dominant, some are recessive “Mendel seven characters” by Mariana Ruiz. Creative Commons CC0 License Structure of DNA Important information that contributed to the discovery of the DNA structure: 1. X-ray diffraction patterns of Rosalind Franklin and Maurice Wilkins 2. Chargaff’s ratio 3. Physical and chemical of constituents: bases, sugar, phosphate Used by James Watson and Francis Crick 1. X-ray diffraction patterns indicated helical structure of the DNA The distinctive “X” tells this is a helix X-ray pattern is regular, so diameter of helix stays the same Vertical distance between bars gives height of one turn Sugar-phosphate backbone deduced to be outside “Experimental setup of Photo 51” by MagentaGreen CC BY-SA 2.0 2. Chargaff’s ratio Chargaff observed that the: number of guanine (G) equals cytosine (C) number of adenine (A) equals thymine (T) DNA base ratio is 1:1 (purines to pyrimidines) Universal (observed in all life forms) Ratio will differ in different species 3. Physico-chemical properties of DNA constituents Molecular structure of the bases, sugar, phosphate Physical (size, shape charge) and chemical properties “GC DNA base pair” and “AT DNA base pair” by Isilanes. Creative Commons CC0 license DNA Double Helix Major Groove Minor Groove “229 Nucleotides-01” by OpenStax College. Creative Commons Attribution 3.0 Unported License A will always pair with T; and G with C; they are complementary to each other.  “0321 DNA Macrostructure” by OpenStax. Creative Commons Attribution 4.0 International License What is a gene? DNA? Gene a DNA codes for a trait  Gene 1 Gene 2 “DNA simple2” by Forluvoft, “Simple chromosome illustration” Creative Commons CC0 license by Prateek Pattanaik. Creative Commons Attribution-Share Alike 4.0 International License "NHGRI human male karyotype" by National Human Genome Research Institute. Creative Commons CC0 License genome TOTALITY of our traits Why was the discovery of Watson and Crick very important? Based on the physical and chemical properties of DNA, Crick and Watson hypothesized a double- helix structure for DNA, much like a twisted ladder. This structure explains: ü How the DNA molecule could replicate or produce identical copies of itself (replication) ü How the information in the gene is expressed (transcription and translation) Copyright CMCSandoval, 2018 Original model of the DNA double helix proposed by Watson and Crick Cavendish Laboratory, University of Cambridge You might ask, why was Rosalind Franklin not included as co-winner of the Nobel prize? This was primarily because Nobel org does not give post- humous awards (Dr. Franklin died before 1962). There are many articles written on this. You can also read the original articles of Dr. Franklin to see for yourself if she should be a co-winner of the Nobel prize. Nonetheless, her contributions to science have been recognized by many. http://www.nobelprize.org/nobel_prizes/medicine/laureates/1962/ Flow of genetic information The information in the gene is transcribed to a complementary single strand called messenger or mRNA; for mRNA, U (uracil) used instead of T (thymine) The mRNA travels from nucleus to cytoplasm and is translated to protein “0328 Transcription-translation Summary” by OpenStax College. Creative Commons Attribution 4.0 International License The information for traits is encoded in the genes Some traits are simple; one gene for one trait ◦ color, selected pest resistance traits Some traits are complex; many genes contribute to the formation of the trait ◦ yield, intelligence  DNA Since the nucleotide sequence of a strand is Parent Strand complementary to that of its partner strand, both strands will bear the same genetic DNA information. This process of DNA replication ensures that the resulting daughter DNA molecules Complementary Complementary Strand Forming are identical to the parental DNA helix. Strand Forming SEMI-CONSERVATIVE “DNA replication split” by Madprime. Creative Commons CC0 1.0 Universal Public Domain Dedication Flow of genetic information “Genetic code” by Madprime. Creative Commons CC0 1.0 Universal Public Domain Dedication “The genetic code wheel” by Genomics Education Programme. Creative Commons Attribution 2.0 Generic License In summary Modified "Central Dogma of Molecular Biochemistry with Enzymes" by Daniel Horspool. Creative Commons Attribution-Share Alike 3.0 Unported License  Part II. Principles of Modern Biotechnology “Modern biotechnology means the application of (a) in vitro nucleic acid techniques, including recombinant DNA technology or direct injection of nucleic acid into cells or organelles (b) Fusion of cells beyond the taxonomic family, that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection.” Cartagena Protocol on Biosafety 2000 Conventional breeding A B (elite line) In conventional breeding, genes of parents are x mixed; desired gene comes with many others which may not have useful or desired traits  Gene of interest (1 page) Genetic engineering Conventional breeding Genome editing- (CRISPR-Cas) Represents one book The genome is like containing a library of books 1000 pages Recombinant DNA technology is a method that allows the combination of genes in a test tube to form a hybrid DNA. Design gene for specific trait Genetic engineering, a type of modern biotechnology, allows the transfer of a specific gene for a desirable trait Basis of Genetic Engineering or Recombinant DNA Technology Why is it possible for a gene from an organism to be introduced and integrated in the genetic material of another organism, and also be expressed? For example, the genes for synthesizing beta-carotene from microorganism and flower can be introduced into the rice plant and be expressed in the rice grain! What is the basis for Genetic Engineering? Cells of different organisms are similar. Thus, a gene transferred to Their cellular constituents are also similar. a new host organism can work and be expressed! They contain the same large and small molecules. They follow the same chemical, physical, and genetic laws. "Simple diagram of plant cell" by domdomegg. Creative "Average prokaryote cell" by Mariana Ruiz Villareal. "Simple diagram of animal cell" by domdomegg. Creative Commons Attribution 4.0 International License Creative Commons Attribution-ShareAlike License Commons Attribution 4.0 International License Tools are available Cutting (splicing) genes at specific sites by using restriction enzymes Ligating DNA fragments using DNA ligase Synthesizing DNA or RNA using DNA or RNA polymerase Multiplying copies of DNA using polymerase chain reaction Cohen (plasmids) of Stanford University and Boyer (restriction enzyme) of UCSF invented recombinant DNA technology, 1972 Let us define some terms... Restriction enzyme – bacterial enzymes that cut DNA at specific sites DNA ligase – enzymes that can form a phosphodiester bond at a single-strand break in DNA Plasmid – double-stranded, circular, autonomously replicating, extra-chromosomal DNA molecules of bacteria Vector – autonomously replicating DNA molecules used to transfer foreign DNA segments between host cells Steps in recombinant DNA technology or genetic engineering 1.) Isolation of gene from source 2.) Modification of gene (if necessary) and prepare gene construct for delivery into target organism 2. Modification of gene and preparation gene construct The gene and its parts promoter structural gene terminator Insert modified gene in appropriate plasmid or vector Bt gene (cry1Ac) Steps in recombinant DNA technology or genetic engineering 1.) Isolation of gene from source 2.) Modification of gene (if necessary) and prepare gene construct for delivery into target organism 3.) Introduce construct to organism (transformation) 4.) Selection of transformants When do we use genetic engineering? the trait to be introduced is not present in the germplasm of the crop the trait is very difficult to improve by conventional breeding methods it will take a very long time to introduce and/or improve such trait in the crop by conventional methods Genetic Engineering of Microorganisms Genetic Engineering of Plants Engineering cinnamon- flavored a b c d e apple f g h i j Genetic Engineering of Animals Current and future applications of modern biotechnology Generations or waves of biotech processes or products Modern Biotechnology Generations Fourth wave Biofuels from Renewable cellulosic resources materials Third wave Factories Industrials, pharmaceuticals Second wave Quality traits Improved nutrition, First wave quality traits Production of health Agronomic traits products in Biotic or abiotic stresses microorganisms and mammalian cells Most of the current products belong to the first wave. "Golden Rice" by International Rice Research Institute (IRRI). Creative Commons CC0 License "Holstein dairy cows" by Scott Bauer. Creative Commons CC0 License The Global Biotechnology Market Global biotech market valued at USD 270.50 billion in 2013; Expected to grow at 12.3% growth rate from 2014 to 2020 Non-food fermentation products } USD 4.9 Billion in 2004 } USD 71.8 Billion in 2021 Adoption of GM Crops 2.5 billion hectares are planted with biotech crops globally A total of 72 countries have adopted biotech crops 29 countries (24 developing countries and 5 industrial countries) planted biotech crops 43 additional countries imported Based on ISAAA report, 2019 GM crop events approved in the Philippines Crop No. of events For propagation Alfalfa 4 Single transformation events Canola 6 Corn MON810 Corn Bt11 Corn TC1507 Corn NK603 Cotton 16 Corn MIR162 Corn MON89034 Eggplant 1 Corn GA21 GR2E Rice Corn 64 Stacked traits/events Potato 11 Corn MON89034 x Corn NK603 Corn TC1507 x Corn MON810 x Corn NK603 and all its intermediates Rice 2 Corn Bt11 x MIR162 x MON89034 x GA21 and all its intermediates Soybean 24 Corn MON89034 x TC1507 x NK603 and all its intermediates Sugarbeet 1 Corn Bt11 x TC1507 x GA21 and all its intermediates Source: ISAAA, 2022 Corn MON810 x NK603 Source: DA-BPI, 2022 GM crop events approved in the Philippines For direct use as food, feed and processing Single transformation events Stacked events Corn (20) Rice (1) Corn (21) Cotton (11) Alfalfa (3) Cotton (4) Canola (2) Potato (3) Soybean (8) Soybean (14) Eggplant (1) Alfalfa (3) Rapeseed (3) Sugarbeet (1) Source: DA-BPI, 2022 Biotech corn — the ffiirst biotech crop in the Philippines By Zabrina J. Bugnosen isaaa photo A filipino farmer tends to drying harvested corn in general Santos city. Biotech corn has been grown in the country since 2003. In December 2002, the Philippines planting season. MON810 changed insect pests like MON810, while was the first country in Asia to all that. After planting MON810, others are tolerant to herbicides approve the commercial planting of farmers realized the benefits of Bt to help farmers manage weeds genetically modified (GM) corn. In corn. They harvested more corn in their farms better. Some its first year of planting, Bt corn was and did not have to buy chemical have additional traits known as Bt corn is first GM crop commercialized in the Philippines The Philippines is the first Asian country to commercialize a genetically modified food crop. In 2002, Bt corn was planted to 10,000 ha. In 2003-2004 to 2007-2008, Bt corn showed consistently higher %ROI, inspite of higher cost of seed. As of 2019, approximately 800,000 ha planted to Bt corn (single trait Bt (insect protected) and HT, (herbicide tolerant) and stacked Bt and HT) The Bt technology Bt used as microbial pesticide for the past 40 years with a history of safe use Bt gene transferred to plant genome; plant produces toxic protein which is pesticidal In the gut of larvae specific toxic protein binds with receptors on gut of target larvae death "Corn borer" by Keith Weller. Creative Commons CC0 License alkaline pH receptor Toxin- Bt protoxin toxin “ulcer-like” receptor The Bt protein in Bt corn is toxic specifically to Lepidoptera dead larva live larva on on Bt corn sweet corn spider lady bug Bt eggplant July 23, 2021 - The Philippine government has approved Bt eggplant for direct use as food, feed or for processing (FFP) [Biosafety Permit No. 21-078FFP] Bt eggplant project led by Dr. Hautea Crossed Bt eggplant developed by Mahyco with Philippine elite lines Field test completed Bt protein affects EFSB worm ONLY Safe for human, farm animals and non-target insects with ABSPII, USAID, UPLB, DA Biotech funds Photos from Dr. DM Hautea, Photos from Dr. DM Hautea and SEARCA BIC SEARCA BIC [email protected] September 2022 https://www.pna.gov.ph/articles/1148086 [email protected] September 2022 Golden Rice produces high levels of beta-carotene in the endosperm phytoene synthase from E. uredovora and phytoene desaturase from daffodil (GR1) and corn (GR2) initiated by Dr. Ingo Potrykus and Dr. Peter Beyer http://www.goldenrice.org/Content2-How/how1_sci.html Golden Rice July 21, 2021 - The Philippine government has approved the COMMERCIAL PROPAGATION of “Golden Rice”. Permit issued by the DA-Bureau of Plant Industry (BPI) based on the rigorous biosafety assessments and recommendation by the Joint Departments – DOST, DA, DENR, DOH and DILG (Joint Department Circular No.1, Series of 2016) https://www.pna.gov.ph/articles/1148037 Courtesy of Dr. Antonio Alfonso, PhilRice [email protected] September 2022 Ringspot virus-resistant papaya Introduction of viral coat protein gene results in overproduction of coat protein RNA and suppression of its synthesis Hawaii Photo courtesy of Dr. Dennis Gonsalves Long shelf-life papaya with PRSV resistance developed at the Institute of Plant Breeding, CAFS, UPLB expression of ACC synthase gene was silence using anti-sense technology Future biotech product Carnation Moondust Carnation (1996) by Florigene delphinidin genes from petunia Available in Au, Canada, USA, UK, Japan, Germany, Holland, Puerto Rico Blue Rose Developed by Suntory & Florigene Used technology for blue carnation-transferred delphinidin genes from pansies https://www.suntory.com/sic/research/s_bluerose/story/ http://www.florigene.com https://www.suntory.com/sic/research/s_bluerose/story/ Transgenic Glofish® gene from jellyfish and anemone injected to eggs of zebra fish developed by Zhiyun Gong of National University of Singapore www.glofish.com AquAdvantage® Salmon developed by Aqua Bounty Farms capability of growing from egg to market size in half the time compared with non-transgenic approved for commercial release in November 2015 http://www.aquabounty.com/ Dr. Arnold Sutterlin with transgenic trout AquAdvantage® Salmon gene construct: growth hormone gene from Chinook salmon under the control of a promoter from another type of fish called an ocean pout. (AP photo/Aqua Bounty) First biopharm drug approved Atryn, an anticlotting drug from GTC Biotherapeutics was approved for release by the European Medicines Agency (June 2006) and US FDA 2009 First biotech product produced in milk of goats (c) GTC Biotherapeutics, now rEVO Biologics Second biopharm drug approved In December 2015, Kanuma was approved by the US FDA; also approved in Europe and Japan Treatment for a rare disease, lysosomal acid lipase (LAL) deficiency, which causes build-up of lipids in cells of various tissues which can lead to liver and cardiovascular diseases. Recombinant lysosomal acid lipase is produced in the egg whites of transgenic chickens. Biopharming: Future products production of pharmaceuticals in crops antibodies, antigens, growth factors, hormones, enzymes, blood proteins and collagen treatment of cancer, HIV, heart disease, diabetes, Alzheimer's disease, kidney disease, Crohn's disease, cystic fibrosis, multiple sclerosis, spinal cord injuries, hepatitis C, chronic obstructive pulmonary disease, obesity and arthritis. List of selected GM products and technologies which are potentially beneficial to the Philippines. Agriculture/Crops Status Other Bt Corn events such as those with resistance to lower ground and upper 1 ground insect pests 2 Rainbow papaya (PRSV-resistant) Available since the 1990s in Hawaii 3 Corn with abiotic stress (drought) tolerance 4 Sugarcane with 30% higher sugar content Indonesia 2020 5 Sugarcane with drought resistance Indonesia 2013 6 Sugarcane with insect protection Brazil (June 2017) Agriculture/Animals 7 AquAdvantage Salmon Approved since 2018; available as food Health 8 Vaccine (traditional and modern) development and production 9 Control of dengue using GM mosquito technology Available since 2002 Industry 10 Cellulosic ethanol Available since mid 2000s 11 Bioethanol Using recombinant yeast Ordonio RL (2022). More modern biotechnologies for applciation in the Philippines and their potential challenges. In: DOST-NCBP. The National Committee on Biosafety of the Philippines (NCBP): Thirty Years of Biosafety Regulations in the Philippines. Department of Science and Technology. National Committee on Biosafety of the Philippines. Manila, Philippines, pp 291-299. Watch the following videos Biotech for Every Juan and Baby Biotech Trip https://www.youtube.com/watch?v=Eoaf1ia2ySU https://www.youtube.com/watch?v=JIj9WbYDNWs Assignment No. 2 Identify two (2) genetically modified organisms in agriculture. 1.) What trait was modified (or traits, if there are multiple)? 2.) Who developed the GMO? 3.) What gene was inserted, deleted or modified (or genes, if there are multiple)? 4.) Is it currently available in the market? If not, at what stage of development or biosafety approval is it currently in?