Genetic Engineering

Basic Principles and Steps involved in Genetic Engineering

1. GENETIC ENGINEERING Dr PULIPATI SOWJANYA Professor & Head Dept. of Pharm. Biotechnology VIGNAN PHARMACY COLLEGE Approved by AICTE, PCI, Affiliated to JNTU Kakinada, Vadlamudi , Guntur Dist. Andhrapradesh-522213 . PRESENTED BY

2. Genetic engineering is the process of using recombinant DNA (rDNA) technology to alter the genetic makeup of an organism. Traditionally, humans have manipulated genomes indirectly by controlling breeding and selecting offspring with desired traits. Genetic engineering involves the direct manipulation of one or more genes. Most often, a gene from another species is added to an organism's genome to give it a desired phenotype. GENETIC ENGINEERING

3. Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing. Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. DEFINITIONS

4. r-DNA technology was introduced in earlier 1960’s, however the techniques were employed mostly in the academic investigations related to the basic mechanisms of cell functions. The advent of classical r-DNA technology provide opportunities for large scale production of therapeutics or human derived proteins and peptides. INTRODUCTION

5. PETER LOBBAN The idea of Recombinant DNA was first proposed by Peter Lobban , a graduate student of Professor Dale Kaiser in the biochemistry department of Stanford university. INTRODUCTION Contd...

6. Herbert Boyer (1936), constructed the first recombinant DNA using bacterial DNA and plasmids. Stanley N. Cohen , received the Nobel Prize in Medicine in 1986 for his work on discoveries of growth factors. INTRODUCTION Contd...

7. The term CLONE means exact copy of the parent. A duplicate or a look like carrying the same genetic signature or genetic map. Cloning is the best application of r-DNA technology and could be applied to something as simple as DNA fragment or larger, sophisticated mammalian species such as humans. CLONING

8. Enzymes Involved in Gene Cloning DNA Polymerase I: It is used for its 5’ 3’ exonuclease activities for synthesizing the complementary strand of DNA or cDNA . Klenow Polymerase: it is the largest fragment of DNA polymerase I possessing 5’ 3’ polymerase and 3’ 5’ exonuclease activities. This is also employed for synthesizing cDNA . T4 DNA Polymerase: This polymerase has 5’ 3’ polymerase and 3’ 5’ exonuclease activites like Klenow polymerase. T 4 DNA polymerase has a marked 3’ 5’ exonuclease activity. It is used for digesting the 3’ overhang formed by restriction endonuclease .

9. Reverse Transcriptase: It is obtained from Avian myeloblastosis virus. It catalyses the synthesis of copy DNA ( cDNA ). Alkaline Phosphatase : This is a zinc containing enzyme when it is isolated from bacteria ( E.coli ) it is known as bacterial alkaline phosphatase (BAP) and when isolated from calf intestine it is known as calf intestine phosphatase (CIP). The enzyme dephosphorylates i.e. removes 5’ terminal phosphate from nucleic acid to generate free 5’ OH group. The dephosphorylation prevents recircularization of vectors so that they remain linearized . Enzymes Involved in Gene Cloning

10. DNA Ligase : It’s action is quite opposite to restriction endonuclease (RE). It is known as molecular suture. It joins two different pieces of DNA. The source of enzyme is T 4 virus or E.coli . The two fragments are brought together by formation of phosphodiester bond between free 5’ – PO 4 group on one strand and 3’ – OH group on the other strand. T4 Polynucleotide Kinase : It possess opposite activity to that of alkaline phosphatase . This enzyme catalyses the addition of a phosphate group at free 5’ OH group of nucleic acid. Terminal deoxynucleotidyl transferase : This enzyme catalyses the addition of a chain of forty or more commonly 20 nucleotides, referred to as ‘tail’, on to the 3’ end of nucleic acids. It is used to add tail in cDNA . Enzymes Involved in Gene Cloning

11. DNase I: It cleaves both ssDNA and dsDNA . It is used to obtain pure RNA. RNase H : It digests RNA. It is used to obtain pure DNA. Methylases : Transfers methyl group at the recognition site. S1 Nucleases : It is a SSDNA endonuclease that cleaves DNA to remove 5’ nucleotides from 5’ end. Enzymes Involved in Gene Cloning

12. STEPS INVOLVED IN GENE CLONING PROCESS

13. Step-1: Gene of Interest 1. Genomic DNA 2. Complementary/Copy DNA ( cDNA ) 3. Chemical Synthesis 4. Mechanical Shearing

14. Isolate whole genomic DNA from organism DNA extraction easily performed using: SDS (detergent) to break up cell membrane and organelles. Salt ( NaCl ) lyses cells and binds the DNA strands together. Proteinase K to digest proteins bound to DNA (essential to remove eukaryotic chromatin). Ethanol ( EtOH ) to precipitate and wash DNA. Water to resuspend and store DNA.

15. Storage - DNA can be stored short-term at room temperature, but is best stored long-term at -80 C or in liquid nitrogen. *Average size of DNA fragments is important for applications involving large regions of DNA sequence/less important for applications involving short regions of DNA sequence.

16. Step 2-Cut DNA with restriction enzymes Restriction enzymes recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond. Cut occurs between the 3 ’ carbon of the first nucleotide and the phosphate of the next nucleotide. Restriction fragment ends have 5 ’ phosphates & 3 ’ hydroxyls. restriction enzyme

17. RESTRICTION ENDONUCLEASES This enzyme is first discovered by Hamellton Smith in Haemophilus influenzae bacteria. This enzyme is also known as ‘ Molecular scissors’. These are used to cut DNA within recognition site. Tools of Recombinant DNA Technology

18. Restriction Endonucleases Type 1 Type 2 Type 3 Cut the DNA away from 1,000 base pairs Cut the DNA with in recognition site. Cut the DNA away from 25 base pairs. TYPES OF RESTRICTION ENDONUCLEASES

19. Actions of restriction enzymes-overview

20. Anneal a short oligo dT (TTTTTT) primer to the poly-A tail. Primer is extended by reverse transcriptase 5 ’ to 3 ’ creating a mRNA-DNA hybrid. mRNA is next degraded by Rnase H , but leaving small RNA fragments intact to be used as primers. DNA polymerase I synthesizes new DNA 5 ’ to 3 ’ and removes the RNA primers. DNA ligase connects the DNA fragments. Result is a double-stranded cDNA copy of the mRNA. Creating a cDNA library

21. Step-2: Insertion into vector Vectors Nucleic acid molecules that deliver a gene into a cell Useful properties Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene Include viral genomes, transposons, and plasmids

22. Step 3-Splice (or ligate) DNA into some kind of cloning vector to create a recombinant DNA molecule Six different types of cloning vectors : Plasmid cloning vector Phage  cloning vector Cosmid cloning vector Shuttle vectors Yeast artificial chromosome (YAC) Bacterial artificial chromosome (BAC) Fosmid cloning vector Zoophaginea Phytophaginea

23. 1. Plasmid Cloning Vectors: Bacterial plasmids , naturally occurring small ‘ satellite ’ chromosome, circular double-stranded extrachromosomal DNA elements capable of replicating autonomously. Plasmid vectors engineered from bacterial plasmids for use in cloning. Features (e.g., E. coli plasmid vectors): Origin sequence ( ori ) required for replication. Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only those cells with the anti-biotic resistance grow in colony). Unique restriction site such that an enzyme cuts the plasmid DNA in only one place. A fragment of DNA cut with the same enzyme can then be inserted into the plasmid restriction site. Simple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ + gene )

24. P BR 322 Ideal vector commonly employed in genetic engineering. It is an artificial vector constructed from plasmid E.coli . It consists 4,363 base pairs. Carries recognition sites for 20 different restriction endonucleases . Carries genes against 2 antibiotics: tet , amp In P BR 322, p stands for plasmid, B and R stand for Bolivar and Rodriguez, names of scientists who constructed this vector and 322 is unique identification number given to the plasmid.

25. P UC 19 Artificially constructed vector. In P UC 19, P stands for plasmid, U and C stand for University of California. It was constructed by J.Messing and J.Veira and 19 is the unique number given for distinguishing it from other plasmids. It contains genes for ampicillin resistance, β - galactosidase ( lac Z) and lac I. It consists of 2680 base pairs.

26. *Cut with same restriction enzyme *DNA ligase Construction of rDNA

27. Engineered version of bacteriophage  (infects E. coli ). Central region of the  chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted. DNA is packaged in phage heads to form virus particles. Phages with both ends of the  chormosome and a 37-52 kb insert replicate by infecting E. coli . Phages replicate using E. coli and the lytic cycle Like plasmid vectors, large number of restriction sites available; phage  cloning vectors useful for larger DNA fragments than pUC19 plasmid vectors. 2. Bacteriophage  cloning vectors:

28. Phage DNA is single stranded Infects only F + cells Comprises of 6402 base pairs A lac Z gene using which transformants can be screened is introduced into a non-coding region of M13. M13 Bacteriophage

29. Features of both plasmid and phage cloning vectors. Do not occur naturally; circular. Origin ( ori ) sequence for E. coli . Selectable marker, e.g. amp R . Restriction sites. Phage  cos site permits packaging into  phages and introduction to E. coli cells. Useful for 37-52 kb. 3. Cosmids or Phagemid 31

30. 4. Shuttle vectors: Capable of replicating in two or more types of hosts.. Replicate autonomously, or integrate into the host genome and replicate when the host replicates. Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells). Example: *Insert firefly luciferase gene into plasmid and transform Agrobacterium . *Grow Agrobacterium in large quantities and infect tobacco plant.

31. 5. Yeast Artificial Chromosomes (YACs) : Vectors that enable artificial chromosomes to be created and cloned into yeast. Features : Yeast telomere at each end. Yeast centromere sequence. Selectable marker (amino acid dependence, etc.) on each arm. Autonomously replicating sequence (ARS) for replication. Restriction sites (for DNA ligation). Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.

32. 6. Bacterial Artificial Chromosomes (BACs) : Vectors that enable artificial chromosomes to be created and cloned into E. coli . Features : Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors. Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects. Like other vectors, BACs contain: Origin ( ori ) sequence derived from an E. coli plasmid called the F factor. Multiple cloning sites (restriction sites). Selectable markers (antibiotic resistance).

33. 7. Fosmid : Based on the E. coli bacterial F-plasmid. Can insert 40 kb fragment of DNA. Low copy number in the host (e.g., 1 fosmid ). Fosmids offer higher stability than comparable high copy number cosmids . Contain other features similar to plasmids/ cosmids such as origin sequence and polylinker .

34. 8. Zoophaginea These are viruses that infect animals. They introduce foreign gene into animal host cells like monkey cells or insects. It uses vectors such as SV40: Simian 40 virus consists of double stranded, circular DNA It is used to introduce foreign gene into monkey cells. (ii) Retrovirus: Introduces foreign DNA into mammalian cells. (iii) Baculovirus : introduces foreign DNA into insect cells.

35. 9. Phytophaginea These are viruses that infect plants and are used to introduce foreign DNA into plants for expression Tomato golden mosaic virus Cauliflower mosaic virus

36. Type of Vector Maximum insert size ( Kbp ) Plasmid vector 15 Bacteriophage Vector 20 Cosmids 45 BAC 300 YAC 2000 Vectors and their maximum hold size for foreign DNA

37. Gene of interest is inserted in vitro into the vector to synthesize rDNA 1. REs generating cohesive ends: If type II RE is used to generate cohesive ends of desired gene and the same RE is used to cut the vector. Then if vector and desired genes are brought together, in vitro annealing occurs. The ends of two DNAs can be joined by DNA ligase at temperature of 4-11 C requiring 12-24 hrs. Step-3: Recombinant DNA

38. 2. RE generating blunt ends: Ligation of desired gee and vector having blunt ends can be brought about by using a high concentration of DNA ligase than that of ligating cohesive ends. It is the T4 DNA ligase that employed rather than Escherichia coli DNA ligase to join blunt ends 3. Homopolymer Tailing: By using RE, if blunt ends are generated then the homopolymer tailing technique is useful for inserting the desired gene into vector. It uses terminal deoxynucleotidyl transferase. Recombinant DNA

39. 4. Linkers and Adapters: It is another technique to convert blunt ends to cohesive ends. Linkers are synthetic oligonucleotides having predetermined recognition and cleavage sites for particular RE that generates cohesive ends on cutting. The linkers are blunt ended on the both sides. First they are phosphorylated using polynucleotide kinase and then ligated to the blunt ended DNA fragment using T 4 DNA ligase . Now the desired DNA fragment is treated with the particular RE, generating the sticky cohesive ends. Then it can be ligated . Recombinant DNA

40. 5. Incompatible cohesive ends generated by use of different REs: If both desired gene and vectors are cleaved with RE to generate cohesive ends incompatible, the pairing up of the bases will not occur. To overcome this cohesive ends are converted to blunt ends in two ways. 1. The protruding end or the overhanging may be removed using S1 nuclease enzyme. 2. The ss can be filled in complementary to the overhang using DNA polymerase enzyme. Recombinant DNA

41. Restriction enzyme nomenclature

42. Step-4: Introduction of rDNA into a host cell Inserting DNA into Cells Goal of DNA technology is insertion of DNA into cell Natural methods Transformation Transduction Conjugation Artificial methods Electroporation Protoplast fusion Injection: gene gun and microinjection 48

43. Transformation Certain bacterial species of genera Streptococcus, Bacillus, Haemophilus , Neisseria and Rhizobium are able to take up the DNA fragments spontaneously under physiological conditions. In some species such as E.coli success rate of transformation is low. Such cells are chemically treated to enhance the ability to take up the foreign DNA. Such treated cells are said to be competent .

44. Transformation Competent cells are prepared by treating with 50mM calcium chloride ice cold solution and then heat shock raising the temperature to 42 ° C to make the movement of foreign DNA into the competent cell.

45. 1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium 3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA 4. Exchange is complete The 4 steps in Transformation

46. Transduction Genetic recombination in which a DNA fragment is transferred from one bacterium to another by a bacteriophage Structure of T4 bacteriophage Contraction of the tail sheath of T4

47. Transduction (Contd...) There are two types of transduction: Generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle. Specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle

48. Seven steps in Generalised Transduction 1. A lytic bacteriophage adsorbs to a susceptible bacterium . 2 . The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes 3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake.

49. Seven steps in Generalised Transduction (cont’d ) 4. The bacteriophages are released. 5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

50. Seven steps in Generalised Transduction (contd) 6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium . 7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

51. Six steps in Specialised Transduction 11. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome . 2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage. Specialised Transduction

52. Six steps in Specialised Transduction (cont’d) 3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid. 4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA.

53. Six steps in Specialised Transduction (cont’d) 5. The bacteriophage adsorbs to a recipient bacterium and injects its genome. 6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

54. Bacterial Conjugation Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium to a recipient bacterium. Often involves a sex pilus. The 3 conjugative processes I. F + conjugation II. Hfr conjugation III. Resistance plasmid conjugation

55. F + Conjugation : Genetic recombination in which there is a transfer of an F + plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus. Other plasmids present in the cytoplasm of the bacterium, such as those coding for antibiotic resistance, may also be transferred during this process. I. F + Conjugation Process

56. The 4 stepped F + Conjugation 1. The F + male has an F + plasmid coding for a sex pilus and can serve as a genetic donor 2. The sex pilus adheres to an F - female (recipient). One strand of the F + plasmid breaks

57. The 4 stepped F+ Conjugation (cont’d) 3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the F+ plasmid enters the recipient bacterium 4. Both bacteria make a complementary strand of the F+ plasmid and both are now F+ males capable of producing a sex pilus. There was no transfer of donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation.

58. II. Hfr Conjugation Genetic recombination in which fragments of chromosomal DNA from a male donor bacterium are transferred to a female recipient bacterium following insertion of an F+ plasmid into the nucleoid of the donor bacterium. Involves a sex (conjugation)pilus.

59. 5 stepped Hfr Conjugation 1. An F+ plasmid inserts into the donor bacterium's nucleoid to form an Hfr male. 2. The sex pilus adheres to an F- female (recipient). One donor DNA strand breaks in the middle of the inserted F+ plasmid.

60. 5 stepped Hfr Conjugation (cont’d) 3. The sex pilus retracts and a bridge forms between the two bacteria. One donor DNA strand begins to enter the recipient bacterium. The two cells break apart easily so the only a portion of the donor's DNA strand is usually transferred to the recipient bacterium. 4. The donor bacterium makes a complementary copy of the remaining DNA strand and remains an Hfr male. The recipient bacterium makes a complementary strand of the transferred donor DNA.

61. 5 stepped Hfr Conjugation (cont’d) 5. The donor DNA fragment undergoes genetic exchange with the recipient bacterium's DNA. Since there was transfer of some donor chromosomal DNA but usually not a complete F+ plasmid, the recipient bacterium usually remains F-

62. III. Resistant Plasmid Conjugation Genetic recombination in which there is a transfer of an R plasmid (a plasmid coding for multiple antibiotic resistance and often a sex pilus) from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus

63. 4 stepped Resistant Plasmid Conjugation 1. The bacterium with an R-plasmid is multiple antibiotic resistant and can produce a sex pilus (serve as a genetic donor). 2. The sex pilus adheres to an F- female (recipient). One strand of the R-plasmid breaks.

64. 4 stepped Resistant Plasmid Conjugation (cont’d) 3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the R-plasmid enters the recipient bacterium. 4. Both bacteria make a complementary strand of the R-plasmid and both are now multiple antibiotic resistant and capable of producing a sex pilus.

65. Artificial methods of inserting DNA into cells: Electroporation Chromosome Electroporation Pores in wall and membrane Competent cell Electrical field applied DNA from another source Cell synthesizes new wall Recombinant cell

66. Artificial methods of inserting DNA into cells: Protoplast fusion C ell walls Protoplast fusion Polyethylene glycol Protoplasts Enzymes remove cell walls Fused protoplasts Recombinant cell New wall Cell synthesizes new wall

67. Artificial methods of inserting DNA into cells: gene gun Gene gun Protoplasts Nylon projectile Nylon projectile Blank .22 caliber shell DNA-coated beads Vent Target cell Plate to stop nylon projectile

68. Artificial methods of inserting DNA into cells: microinjection Microinjection Target cell Suction tube to hold target cell in place Target cell’s nucleus Micropipette containing DNA

69. Step-5: Identification & Isolation of Transformed cells The transformed cells are identified on the basis of some selective property that has been acquired by the transformed cells. Most frequently markers coding for specific antibiotic resistance are used. Resistance against antibiotics, heavy metals Production of antibiotics, bacteriocins , enterotoxins , H 2 S Metabolism/degradation of aromatic compounds, sugars, haemoglobin . Induction of plant tumour

70. Overview of rDNA technology Bacterial cell Bacterial chromosome Plasmid Gene of interest DNA containing gene of interest Isolate plasmid. Enzymatically cleave DNA into fragments. Isolate fragment with the gene of interest. Insert gene into plasmid. Insert plasmid and gene into bacterium. Culture bacteria. Harvest copies of gene to insert into plants or animals Harvest proteins coded by gene Eliminate undesirable phenotypic traits Produce vaccines, antibiotics, hormones or enzymes Create beneficial combination of traits

71. Applications of Recombinant DNA Technology Environmental Studies Most microorganisms have never been grown in a laboratory Scientists know them only by their DNA fingerprints Allowed identification of over 500 species of bacteria from human mouths Determined that methane-producing archaea are a problem in rice agriculture

72. Applications of Recombinant DNA Technology ( Contd …) Pharmaceutical and Therapeutic Applications Protein synthesis Creation of synthetic peptides for cloning Vaccines Production of safer vaccines Subunit vaccines Genes of pathogens introduced into common fruits and vegetables Injecting humans with plasmid carrying gene from pathogen Humans synthesize pathogen’s proteins

73. Applications of Recombinant DNA Technology ( Contd …) Pharmaceutical and Therapeutic Applications Genetic screening DNA microarrays used to screen individuals for inherited disease caused by mutations Can also identify pathogen’s DNA in blood or tissues DNA fingerprinting Identifying individuals or organisms by their unique DNA sequence

74. GOLDEN RICE : A Recombinant variety of rice that has been engineered to express the enzymes responsible for β carotene synthesis. AGRICULTURE: Growing crops of your choice. Pesticide resistant crops. Fruits with attractive colours. All benign grown in artificial conditions. Applications of Recombinant DNA Technology ( Contd …)

75. PHARMACOLOGY: Artificial insulin production. Drug delivery to target sites. MEDICINE: Gene therapy Antiviral therapy Vaccination Synthesising clotting fact ors Applications of Recombinant DNA Technology ( Contd …)

76. Applications of Genetic Engineering in Medicine A) Insulin production: Human insulin produced through GE since 1982. Human insulin gene inserted into the bacterium E.coli to produce synthetic "human" insulin, for the treatment of insulin-dependent diabetes. In past, insulin was obtained from a cow or pig pancreas, that has many problems. B) Producing human growth hormones: to treat growth retardation (dwarfism). C) Producing Follistim injection: (contains the FSH hormone) for treating infertility. D) Other biopharmaceuticals under development through genetic engineering, include: anti-cancer drug and a possible vaccine for AIDS, malaria, etc.

77. E) Making human albumin, anti-hemophilic factors and many other drugs. F) GE vaccines may be useful to prevent diseases that have resistant to traditional vaccination , including HIV, tuberculosis…etc. G) Gene therapy has been successfully used to treat Chronic lymphocytic leukemia (CLL) and Parkinson's disease. H) Gene therapy is also being tested as a treatment for cystic fibrosis, skin cancer, breast cancer, brain cancer, and AIDS. However, most of these treatments are only partially successful. The major reasons for these failures is inefficient vectors  Applications of Genetic Engineering in Medicine

78. ADVANTAGES : Provide substantial quantity. No need for natural or organic factors. Tailor made product that you can control. Unlimited utilisations. Cheap Resistant to natural inhibitors.

79. DISADVANTAGES: Commercialised and became big source of income for business man. Effects natural immune system of the body. Can destroy natural ecosystem that relies on organic cycle. Prone to cause mutation that could have harmful effects. Major International concern: Manufacturing of biological weapons such as botulism and anthrax target humans with specific genotype. Concern of creating super human care.

80. Genetic Engineering https://www.youtube.com/watch?v=BK12dQq4sJw

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