1. Introduction to Genetic Engineering.pptx

Bioengineering

1. 1 Introduction to Genetic Engineering and Biosafety 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 1

2. Introduction 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 2 What is Genetic Engineering?, Definitions, Purpose…. Chronological advancement of genetic engineering Steps involved in genetic engineering Applications of genetic engineering

3. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 3 1 I n t r o du c t i o n Genetic Engineering The simple addition, deletion, or manipulation of a single trait in an organism to create a desired change

4. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 4 Genetic Engineering What you need to know? Manipulation and alteration of genes Three applications: one plant, one animal, one micro-organism Process involving isolation, transformation, and expression

5. Genetic Engineering Is artificially copying a piece of DNA from one organism and joining this copy of DNA into the DNA of another organism www.clipartguide.com 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 5

6. 6 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Genetic Engineering Modern biotechnology relies on newer techniques, such as genetic engineering, to incorporate genetic material from one living organism into another. The DNA is cut from the donor cell and inserted into the DNA of another organism (vector) e.g. bacterium. The bacterium will then manufacture the protein coded for by the gene. Products of biotechnology include human insulin, and enzymes used in laundry detergents and cheese-making. More recently, the use of biotechnology has led to new pesticide products that control a variety of pests.

7. 7 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Purpose of Genetic Engineering It allows genes from one organism to be inserted into a cell of a different organism of a different species (that DNA from different organisms can be combined). Examples: Human genes can be inserted into a bacterium (Bacteria can be engineered to produce human proteins) Human genes can be inserted into cells from other animals Bacterium genes can be inserted into plant cells

8. 8 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Genetic Engineering The altered DNA is called recombinant DNA Recombinant DNA is joined to other unrelated DNA in the organism . This is called gene splicing . Segments of a gene are taken out and replaced by different genes

9. 9 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Transgenic Organisms Transgenic Organisms: Organisms whose DNA is altered by genetic engineering. Genetic material changed by other than random natural breeding through gene transfer Gene transfer: moving a gene from one organism to another.

10. 10 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU What Transgenic means? 'Trans-' means 'crossing from one place to another‘ The '-genic' means genes So it means that bits of genes from different living things have been bolted together and spliced into another organism to make a new one which does something which the scientists want it to do.

11. Examples of Transgenic Organisms 11 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU GMO- genetically modified organism For example Plants that resists a particular type of weed killer Sheep which makes some special substance in its milk.

12. Transferring a desired gene from one organism to another and the one which receives the foreign gene is known as transgenic organism or genetically modified organism (GMO) . 12 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

13. 13 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Chronological advancement of genetic engineering

14. The beginnings 1869 DNA isolated 1944 DNA proved to be the hereditary material 1953 DNA structure determined. 14 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

15. Working with DNA 1961 DNA can be split on heating (dis-socation) and stuck back together again on cooling (re- annealing) , DNA hybridization possible 1962 The first restriction endonucleases (molecular scissor)discovered. H i n d I I I 15 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

16. Working with DNA 1963 The genetic code was discovered to be a universal code. S cientists are now able to predict characteristics by studying DNA. This leads to genetic engineering, genetic counseling. 1967 DNA ligase discovered Molecular glue Fragments of DNA can be stuck together. DNA Ligase 16 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

17. 17 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Recombinant DNA technology 1973 DNA cloning carried out on bacteria Gene identified Cut with restriction enzyme Spliced into a plasmid using ligase Plasmid reintroduced into a bacterium Gene copied whenever the bacterium divides Non-bacterial gene can be expressed in the bacterium.

18. Recombinant DNA technology 1977 Rapid sequencing of DNA developed. 18 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

19. Recombinant DNA technology 1982 Transgenic mice and fruit flies produced. Transgenic mice express enhanced green fluorescent protein 19 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

20. Recombinant DNA technology 1983 Polymerase chain reaction invented 1985 Genetic fingerprinting developed. Kary Mullis Sir Alec Jeffreys 20 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

21. M ed i c i n e 1989 Cystic fibrosis gene cloned and sequenced 1990 Trials for gene therapy begin. 21 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

22. Agriculture 1994 Genetically modified organisms for food. Flavr Savr TM tomatoes 22 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

23. 23 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Human Genome Project 1990-2003 The human genome worked out Goals of the human genome project identify all the approximately 20,000-25,000 genes in human DNA determine the sequences of the 3 billion chemical base pairs that make up human DNA store this information in databases improve tools for data analysis transfer related technologies to the private sector address the ethical, legal, and social issues (ELSI) that may arise from the project.

24. The history of genetics since 1900. Shaded areas represent the periods of major development in each branch of the subject. 24 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

25. 25 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Genomics 2015: Over 13700 genomes for different species have been sequenced NCBI database Genome http://www.ncbi.nlm.nih.gov/genome/ Of which over 2400 Eukaryotes sequenced About 587 of these are animals Over 7300 prokaryotes (bacteria and archaea) have been sequenced.

26. 26 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Genomics and Proteomics The field of genomics deals with the DNA sequence, organization, function, and evolution of genomes Proteomics aims to identify all the proteins in a cell or organism including any posttranslationally modified forms, as well as their cellular localization, functions, and interactions Genomics was made possible by the invention of techniques of recombinant DNA, also known as gene cloning or genetic engineering

27. Structural Genomics 27 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

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29. 29 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Genetic Engineering In genetic engineering, the immediate goal of an experiment is to insert a particular fragment of chromosomal DNA into a plasmid or a viral DNA molecule This is accomplished by breaking DNA molecules at specific sites and isolating particular DNA fragments DNA fragments are usually obtained by the treatment of DNA samples with restriction enzymes Cloning from mRNA molecules depends on an unusual polymerase, reverse transcriptase , which can use a single-stranded RNA molecule as a template and synthesize a complementary DNA ( cDNA )

30. 30 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU cDNA Cloning The resulting full-length cDNA contains an uninterrupted by introns coding sequence for the protein of interest If DNA sequence is known at both ends of the cDNA for design of appropriate primers, amplification of the cDNA produced by reverse transcriptase is possible by reverse transcriptase PCR ( RT-PCR)

31. 31 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Bioinformatics Rapid automated DNA sequencing was instrumental in the success of the Human Genome Project , an international effort begun in 1990 to sequence the human genome and that of a number of organisms However, a genomic sequence is like a book using an alphabet of only four letters, without spaces or punctuation. Identifying genes and their functions is a major challenge The annotation of genomic sequences at this level is one aspect of bioinformatics , defined broadly as the use of computers in the interpretation and management of biological data.

32. 32 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Functional Genomics Genomic sequencing has made possible a new approach to genetics called functional genomics , which focuses on genome-wide patterns of gene expression and the mechanisms by which gene expression is coordinated DNA microarray (or chip ) - a flat surface about the size of a postage stamp with up to 100,000 distinct spots, each containing a different immobilized DNA sequence suitable for hybridization with DNA or RNA isolated from cells growing under different conditions DNA microarrays are used to estimate the relative level of gene expression of each gene in the genome

33. DNA microarray 33 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

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35. 35 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU Reverse Genetics Mutation has traditionally provided the raw material needed for genetic analysis. The customary procedure has been to use a mutant phenotype to recognize a mutant gene and then to identify the wildtype allele and its normal function Recombinant DNA technology has made possible another approach, often called reverse genetics , in which wildtype genes are cloned, intentionally mutated in specific ways, and introduced back into the organism to study the phenotypic effects of the mutations

36. Transgenic Animals Germ-line transformation involves the insertion of genes into the reproductive cells of an organism, which permanently alters the genetic content of the individual and all offspring = transgenic animals Transgenic animals are used to study the functions of specific genes in development or disease processes 36 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU

37. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 37 Stages involved in Genetic Engineering

38. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 38 Stages involved in Genetic Engineering Isolation of gene of interest and selection of vector Cutting gene of interest and vector with the restriction enzyme Ligation and Insertion in to the host cell Transformation Expression

39. Genetic probe 1. Isolation (a) Isolation of a specific gene from donor e.g. human Cells broken open Genetic probe added Reveals position of the gene of interest Position of gene of interest 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 39 Donor DNA

40. 1 . I s o l a t i o n Pla s mid (b) Isolation of plasmid from a bacterial cell Bacterial cell 3/13/2020 Genetic Engineerin w g w a w nd .s B c io i. s s a d fe s t u y_ .e M d u u lugeta K_ASTU 40

41. 2. Cutting Restriction enzymes act as molecular scissors and cut DNA at specific sites called restriction sites Restriction site R e striction site R e striction ezymes 3/13/2020 Genetic C E li n p g a i r n tg e u e i r d i e n . g co a m nd Biosafety_Mulugeta K_ASTU 41

42. 2. Cutting 42 Plasm i d Restriction site Restriction site Donor DNA R e st r i ction enzymes 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU © Biology Support Service 2007

43. Cu t t i n g Donor DNA Sticky Ends 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 43 Pl a s mid

44. DNA Ligase Ligation –re-joining cut fragments of DNA and forming artificial recombinant molecules 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 44

45. 3. Ligation and Insertion 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 45

46. 4. Transformation Recombinant DNA introduced into bacterial cell Bacterial chr o mos o me B a cteri a l cell Recombinant DNA 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 46

47. Summary of steps Donor DNA Pl a s mid 1. Cut with restriction enzymes Donor DNA Stic k y Ends 2. Ligase bonds sticky ends together Recombinant DNA 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 47

48. 5 . E x p r e s si o n Bacterial cell reproduces by Binary Fission Bacterial cell produces the polypeptide coded for by the donor DNA 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 48

49. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 49 E x p r e ss i o n Expression is getting the organism with the recombinant DNA to produce the desired protein When the protein is produced in large amounts it is isolated and purified

50. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 50 Applications of Genetic Engineering

51. Plant Application Golden Rice – a possible solution to Vitamin A deficiency. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 51

52. “ Golden Rice ” “Golden Rice” is a genetically modified crop that was developed by Swiss and German scientists in 1999 . This breed of rice was engineered to produce higher levels of beta-carotene, the precursor of Vitamin A in the human body. Increased levels of this substance give the rice its characteristic “golden” color. Millions of people around the world suffer from vitamin A deficiency (VAD), with the majority living in developing countries. Advanced VAD can lead to blindness. Pregnant women and children are especially vulnerable. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 52

53. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 53 P l a n t s Vitamin A in Rice - The gene which produces vitamin A was taken from daffodils and put into rice to help prevent blindness Weedkiller resistant crops - Weeds die but the crops survive

54. A tomato implanted with a gene from E. coli . A tomato that will not soften while ripening on the vine. The transgenic tomato would allow tomatoes to be shipped safely, keep their color, and have their natural flavors . Increased shelf life. First Commercially Genetically Modified Food Flvr Savr 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 54

55. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 55 Molecular Farming The manufacturing of valuable pharmaceutical or industrial recombinant proteins using plant-based production systems Molecular farming is turning plants into factories in a sense that we use the molecular machinery of plants in order to produce a desired protein with economic interest.

56. Molecular Farming – Genetic Transformation Candidate gene Structure gene Plant Expression Vector I n t r o d u ct i on in the plant Transgenic Plant Plantation H a r v e s t Extraction and Purification Metabolites 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 56

57. Examples - Plant-based Vaccines Plant-based human vaccines in clinical trials Instead of using this… You could use this… Adapted from Ther Adv Vaccines (2015), 1-16 Safety – Don´t carry an alive pathogen (only the capsid protein) Storage and delivery – Can be stored at room temperature and orally delivered, which is very important for developing countries Pathogen or disease Antigen Pla n t Ex p r e ss i on system C lini c a l trial Reference Enterotoxigenic E.coli LTB Potato and Maize Transgenic Phase I Tacket et al. , 1998, 2004 Norovirus Capsid protein Pota t o Transgenic Phase I Tacket et al. , 2000 Hepatite B virus Viral major surface protein Lettuce and Potato Transgenic Phase I Kapusta et al ., 1999 and Thanavala et al., 2005 Rabies virus Glycoprotein and nu cle o pr otein S p i n ach Viral vector Phase I Yusibov et al., 2002 Influenza virus (H5N1; 2009 pandemic) HA Nicotina b en tha mi an a L a u n ch vector Phase I Cummings et al, 2014 Cholera CTB Rice Transgenic Phase I Nochi et al. , 2009 and Yuki et al., 2013

58. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 58 Antisense technology Used to produce the Flavr-Savr tomato in 1994. Enzyme polygalacturonase (PG) breaks down structural polysaccharide pectin in wall of a plant. This is part of the natural decay process in a plant Monsanto identified the gene than encodes the enzyme and made another gene that blocked the production of the enzyme .

59. Antisense t echnology 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 59

60. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 60 Applications of Plant Genetic Engineering in agriculture Crop Improvement Modification of plant nutritional content: increasing the vitamin A content of plants Genetically Engineered Traits: 1.Herbicide Resistance 2.Insect Resistance 3.Virus Resistance 4.Altered Oil Content 5.Delayed Fruit Ripening Biotech Revolution: Cold and Drought Tolerance and Weather-Gard Genes Genetically Engineered Foods 1.Soybeans Corn Cotton 4.Other Crops

61. Genetically modified crops Insect R e sis t ance He r bicide T olera n ce Drought resis t ance 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 61 Yield increase Nutrient En h ance m ent Virus T olera n ce Advantages of GM Crops

62. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 62 Gene Pharming Gene pharming is a technology that scientists use to alter an animal's own DNA, or to splice in new DNA, called a transgene, from another species. In pharming, these genetically modified (transgenic) animals are mostly used to make human proteins that have medicinal value. The protein encoded by the transgene is secreted into the animal's milk, eggs or blood, and then collected and purified. •

63. Tracy -the Sheep One of the first mammals engineered successfully for the purpose of pharming was a sheep named Tracy, born in 1990 and created by scientists led by British developmental biologist Ian Wilmut at Roslin Institute in Scotland. Tracy was created from a zygote genetically engineered through DNA injection to produce milk containing large quantities of the human enzyme alpha-1 antitrypsin, a substance used to treat cystic fibrosis and emphysema 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 63

64. Applications (Micro-organisms) Production of humulin www.healthtap.com Used by diabetics 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 64

65. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 65 Micro organisms Bacteria can make human insulin This prevented many diabetics from getting an allergic reaction to animal insulin Bacteria make interferon which can fight virus infections and some cancers

66. Vaccines Genetically engineered microbes can be used to produce the antigens needed in a safe and controllable way. The use of genetically modified yeast cells to produce a vaccine against the hepatitis B virus has been a major success story. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 66

67. Gene Therapy It involves modifying human DNA either to repair it or to replace a faulty gene. The idea of gene therapy is to overcome the effects of a mutation which causes a genetic disease . Cystic fibrosis is the best known disease where gene therapy has been tried. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 67

68. 3/13/2020 Genetic Engineering and Biosafety_Mulugeta K_ASTU 68 Diagnostic Tests Genetic engineering can produce very specific and sensitive diagnostic tests for many diseases, using engineered proteins. This new technology is also opening up novel ways of delivering medicines to specific targets.