8. Genetic enginering basic of modern biotecnology.pptx

GMO as a product of biotechnology

1. basic of modern biotecnology ILMU KEALAMAN DASAR

2. Biotechnology

3. Biotechnology Is a field of applied biology which involves the use of living organisms or other biological system in manufacture of drug or other product or environmental management Louis Pasteur known as a father of biotechnology

4. Field of biology that involved Biotechnology Microbiology Cell biology Biochemistry Genetic

5. Biotechnology Traditional Biotechnology modern Biotechnology

6. Conventional/traditional biotechnology Biotechnology which involed of microorganism (fungi bacteria) , biochemical process and mutation of genes that occured naturally Reason of using bacteria or fungi: Both are very small and easy to grow in the lab, reproduce quickly No ethical issues of the usage of those both. Share the same kind of genetic material (DNA) Bacteria contain little loops (circular) of DNA  plasmid easily to be transferred to the other cells (organism)

7. YEAST

8. The use of yeast Yeast is a microorganism which can respire anaerobically to release carbon dioxide Anaerobic respiration equation glucose  alcohol + CO 2 + energy Used to make : Bread Biofuel (bioethanol)

9. The use of yeast Making  starch of the flour used by yeast to grow Carbon dioxide helps the bread to rise Bread

10. The use of yeast Making  Ethanol is also released during this reaction Biofuel

11. Father of biotechnology Proved that fermentation done by microorganism (1857-1876)

12. Production of penicillin

13. Modern biotechnology Biotechnology which invole of manipulation and genetic engineering beside biochemistry and microbiology

14. Genetic engineering Genetic engineering is a technique used to transfer genes from one organism to the other organism Plasmid of bacteria is needed to transfer the genes. Plasmid funtioned as a vector (carrier) of genetic material plasmid is DNA molecules that are circular shape and found in most species of bacteria that exist apart from the bacterial chromosome

15. History It began around 50 years ago based on the research of Dr. Paul Berg from university of Stanford They found the enzymes work to cut the DNA and tto glue the DNA and The hybrid of DNA (DNA which is modified and contain DNA of other organism) called recombinant DNA

16. Enzymes involved on genetic engineering Restriction enzymes are protein functioned to cleave (cut) DNA at spesific sequence that it recognizes Ligase enzymes are the enzyme functioned to glue two or more fragments of DNA

17. DNA Recombinant combining genes from two different species Manufacturing recombinant DNA molecules requires : Restriction enzymes  to cut donor and recipient DNA at the same sequence DNA to carry the donor DNA  cloning vectors Recipient cells (bacteria)

18. Bacteria involvement Some bacteria might be used is genetic engineering Escherichia coli involve in production of synthetic insulin hormon Agrobacterium tumefaciens involve in genetic plant modification

19. Restriction enzymes Restriction endonucleases  enzymes from bacteria which recognise and break down the DNA.  to cut DNA into smaller pieces Cut in the back bone of DNA  how it work? Bind on specific site of specific sequence bases Cut either straight or blunt end

20. Common Restriction Enzymes Fig. 13.8

21. Modifying plasmid of bacteria the other enzyme needed  DNA ligase Plasmid can also made artificially, example pUC group of plasmid, because: a low molecular mass an origin of replication so they can be copied several single target sites for different restriction enzymes in a short length of DNA called a polylinker one or more marker genes, allowing identification of cells that taken by plasmid

22. Fig. 13.11a-d Cloning DNA in Plasmid Vectors

23. Fig. 13.11e-g

24. Plasmid Used to Carry DNA Fragments = Vectors Fig. 13.10

25. Table 19.2

26. EcoRI EcoRI EcoRI EcoRI 4.0 kb 2.0 kb 3.0 kb Problem: How to get the 2.0 kb piece to subclone into vector Randomly Isolate specific fragment Shotgun cloning

27. DNA RECOMBINANT The Procedures Selecting cells where the genetic material includes any foreign DNA. Selecting cells that received the gene of interest. Stimulating transcription of the foreign gene and translation of its protein product. Collecting and purifying the desired protein

28. Steps in cloning a single piece of DNA 1. Appropriate restriction sites 2. Cut vector and foreign DNA with RE 3. Run on gel to separate fragments 4. Isolate specific fragment 5. Ligate with cut vector 6. Transform host bacteria. Selection. 7. Grow up colonies. 8. Isolate plasmid DNA. 9. Cut with RE to confirm presence of foreign DNA. 10. Run on gel to identify recombinant plasmids.

29. INSULIN Production

30. DNA Replication – In-Vivo Strand “unzips”, hydrogen bonds between base pairs are broken. Sequence of bases on strand serve as template to which complementary bases are added. When process is complete 2 identical DNA molecules are formed.

31. DNA Replication – In-Vitro Two steps Denaturation – can use heat or alkaline solutions to break bonds and separate the two strands, if heat is used term is called “melting”. Annealing – strands cool and complementary strands spontaneously rejoin. This process is exploited in the laboratory.

32. DNA Sequencing

33. Target Amplification In-vitro systems for enzymatic replication of target molecule to detectable levels. Allows target to be identified and further characterized. Examples: Polymerase chain reaction (PCR) Transcription mediated amplification (TMA) Strand displacement amplification (SDA) Nucleic acid sequence-based amplification (NASBA)

34. Polymerase Chain Reaction Capable of amplifying tiny quantities of nucleic acid. Cells separated and lysed. Double stranded DNA separated into single strands (denatured). Primers, small segments of DNA no more than 20-30 nucleotides long, added. Primers are complementary to segments of opposite strands of that flank the target sequence. Only the segments of target DNA between the primers will be replicated. Each cycle of PCR consists of three cycles : Denaturation of target DNA to separate 2 strands by heating. Annealing step in which the reaction mix is cooled to allow primers to anneal to target sequence Extension reaction in which primers initiate DNA synthesis using a DNA polymerase. These three steps constitute ONE thermal cycle Each PCR cycle results in a doubling of target sequences and typically allowed to run through 30 cycles, one cylce takes approximately 60-90 seconds.

35. Taq Taq polymerase ("Taq pol") is a thermostable polymerase isolated from thermus aquaticus, a bacterium that lives in hot springs and hydrothermal vents. "Taq polymerase" is an abbreviation of Thermus Aquaticus Polymerase. Often used in polymerase chain reaction, since it is reasonably cheap and it can survive PCR conditions.

36. Polymerase Chain Reaction http://www.dnalc.org/resources/animations/pcr.html

37. Polymerase Chain Reaction

38. Polymerase Chain Reaction Sample with all test components mixed together. Put in thermocycler which cycles the temperature for each stage of the reaction. Electrophorese to separate and identify.

39. Markers The antibiotic resistance genes added to the plasmids along with the human insulin gene act as markers. They make it possible to identify the bacteria that have taken up the gene. There is a concern that using antibiotic resistance genes as markers could increase the likelihood of the development of populations of harmful bacteria that are resistant to antibiotics. Today, most common markers used are genes that code for the production of fluorescent green protein. The gene for this protein can be inserted along with the desired gene. Cells that fluoresce green are therefore likely to have taken up the desired gene.

40. Gel Electrophoresis Electrophoresis is way of separating different molecules of different length (separating DNA fragments of varying sizes ranging from 100 bp to 25 kb) To determine the sizes of DNA fragments To determine the presence or amount of DNA

41. Principle Gel Electrophoresis powerful separation method frequently used toanalyze DNA fragments generated by restriction enzymes convenient analytical method for determining the size of DNA molecules in the range of 500 to 30,000 base pairs employs electromotive force to move molecules through a porous gel

42. The movement of charged molecules within the gel

43. Gel Electrophoresis Cut DNA by restriction enzymes Place on agarose gel Apply current Fragments travel toward anode Short fragments travel further Visualise DNA with UV light

44. Variable number of tandem repeat The full genetic profiles of any two individuals  different except identical twins, but, Human genes are the same from person to person, DNA typing relies on the stretches of DNA that tend to differ among different people. The repeated sequences themselves are usually the same from person to person  known as Variable Number of Tandem Repeats VNTR same as Shorts Tandem Repeat the difference being that in a VNTR, the repeated sequence is longer — about 10-100 base pairs long.

45. Microarray A DNA microarray is composed of pieces of DNA ranging from 20-5000 base pairs concentrated into specific areas on a solid support such as a glass or silicon (2 cm 2 ) Thousands to hundred thousands spots per inch  each hold million copies DNA Each spot of DNA called as probe, represent single gene. Each spot may contain few million copies of identical probe molecules Microarray  called as DNA chips, gene chips, DNA arrays, biochips The use of microarrays for gene expression profiling was first reported in 1995 Complete eukaryotic genome ( Saccharomyces cerevisiae ) on a microarray published in 1997

46. Principle of Microarray Principle of microarray  hybridization Samples labeled using fluorescent At last 2 samples hybridized to chip Fluorescently labeled target sequences that bind to a probe sequence generate a signal The signal depends on : hybridization condition ( temperature) and washing after hybridization

47. Scanning the array Laser scanner Ccd scanners

48. The use of Microarray to compare the genes present in two different species to detect which genes are being expressed at any specific time in each cell in the body, example, the genes that are expressed in a cancer cell are different from those active in non-cancerous cells

49. Bioinformatic

50. Bioinformatics “bioinformatics”  first used in the mid-1980s in order the application of information science and technology in the life sciences. Bioinformatics  biological data with computer technology and statistics active research field, began with handling and analysis of DNA sequences. Bioinformatics  genomic functions to determine the role of the sequence in living cell (protein, its description and functions and comparative genomic.  database hold gene sequences, sequences of genomes, amino acid sequences of protein and it structures Bioinformatics may be able to provide the needed quantification for the vast tracts of biology

51. Bioinformatics  Human genome project Produced the human genome sequence And now provides us an unparalleled opportunity to apply new knowledge, technologies, and approaches to health care. Bioinformatics deals with any type of data that is of interest to biologists : DNA and protein sequences, Gene expression (microarray) More data in when we attempt to : relate to structure, physiology, relate to disease, relate to variation

52. Some research of Bioinformatics The Human Genome Project -- old news, 6 years ago • International HapMap Project -- www.hapmap.org • The 1000 Genomes Project – www.1000.genomes.org • Encyclopedia of DNA Elements ( ENCODE ) Project • The Cancer Genome Atlas (TCGA) • Human Microbiome Project (HMP) – www.hmpdacc.org • The eMERGE (Electronic Medical Records and Genomi

53. Genetic engineering and medicine

54. human growth hormone thyroid stimulating hormone factor VIII – a blood clotting protein. advantages in using bacteria, yeasts and cultures of mammalian cells to produce these proteins have simple nutritional requirements large volumes of product are produced the production facilities do not require much space the processes can be carried out almost anywhere in the world disadvantage of using bacteria to produce human proteins is that bacteria do not modify their proteins in the same way that eukaryotes Genetic engineering and medicine

55. Genetic engineering and medicine factor VIII – a blood clotting protein via genetically modified hamster  inserting human gene into kidney and ovary cells enzyme adenosine deaminase (treat severe combined immunodeficiency disease)  genetically larva of cabbage looper moth caterpillar human antithrombin is produced by goats human alpha-antitrypsin is produced by sheep

56. Genetic screening

57. Amniocentesis puncture to remove fluid withdrawing some of the amniotic fluid (10 to 30 mL) that bathes the developing fetus and analyzing the fetal cells and dissolved substances to test certain genetic disorders  down syndrome, hemophilia, Tay -Sachs disease, sickle-cell disease done at 14–18 week the position of the fetus and placenta is first identified using ultrasound and palpation the skin is prepared with an antiseptic and a local anesthetic is given, a hypodermic needle is inserted through the mother’s abdominal wall and into the amniotic cavity within the uterus

58. viruses (retroviruses or lentiviruses)  small spheres of phospholipid called (liposomes)  vectors

59. Gene Therapy

60. SCID severe combined immunodeficiency, do not have an effective immune system  extremely vulnerable to any form of infection. ADA  gene  instruction to produce enzyme adenosine deaminase Highest levels of adenosine deaminase  lymphocytes  protect against pathogen Lymphocytes  lymphoid tissue  thymus gland Function of the adenosine deaminase enzyme is to eliminate a molecule called deoxyadenosine  generated when DNA broken Adenosine deaminase converts deoxyadenosine, which is toxic to lymphocytes

61. SCID Case : 1 in 50.000 – 100.000 birth Gene therapy involving retroviruses or lentiviruses (RNA virus) or small spheres of phospholipid called liposomes keeping children alive  bring them up in a completely sterile environment, no direct contact  rarely live into teenager Alternative treatment  bone marrow transplant (suitable donor)  rejection (transplanted marrow attack the patient’s cells) ; donor cells infected by virus

62. SCID and gene therapy inserting a healthy gene into the DNA using a vector such as a specially modified virus  develop functioning immune system by enable fight antigen by antibody Leukaemia like symptom might appear

63. Sickle cell disease and gene therapy Caused by a mutation in a single gene affects the formation of one of the two types of protein chain which make up haemoglobin  change the shape if haemoglobin 2017  French scientists announced that they had reversed the progress of sickle cell disease in a teenage Therapy  bone marrow stem cells Bone marrow stem cells  genetically modified them using a viral vector so they could make functioning haemoglobin , and replaced the stem cells in the patient

64. Genetic engineering and gene silencing silencing, specific genes can be shut down so that they no longer produce a rogue protein. by injecting nematode worms with a double-stranded piece of RNA which corresponds to a particular gene, they can block its action The potential for this in the treatment of diseases like cancer and AIDS 2010 the first disease actually prevented using gene silencing was a respiratory infection caused by a virus known as RSV nasal spray containing RNA fragments to silence one of the viral genes

65. Cystic fibrosis Cystic fibrosis is a genetic disorder in which abnormally thick mucus is produced in the lungs and other parts of the body Cilia can not push up from the lungs  bacteria breed in it Thick mucus  block pancreatic duct (stop the enzyme), thick secretions block ducts in the reproductive system (sterile) The cause  recessive allele of the gene that codes for a transporter protein (CFTR) CFTR  cell surface membranes of alveoli  allow chloride ion to pass out the cell

66. cells lining the airways and in the lungs pump out chloride ions (Cl−) through the channel in the cell surface membrane formed by CFTR h igh concentration of chloride ions outside the cells (reduce water potential) -  water move out by osmosis  mucus mixed, thin and easy removed CFTR gene  chromosome number 7 (250 000 bases) Mutation  deletion of 3 bases (1 AA)  stop codon Gene therapy  If the normal dominant allele could be inserted into cells in the lungs, the correct CFTR should be made drug (PTC124) has been found to allow translation to just keep going across this stop codon Cystic fibrosis - CFTR

67. Somatic and germ cell gene therapy Gene therapy involves introducing a ‘correct’ allele into a person’s cells as a treatment for a genetic disease Placing the allele in body cells or insert the allele into germ cells

68. Genetic technology and agriculture Brassica napus (seed rape)  source of vegetable oil which is used as biodiesel fuel, as a lubricant and in human and animal foods Natural rape seed oil contains substances (erucic acid and glucosinolates )  undesirable in oil Canola (Canadian oilseed low acid)  hybrid, bred in Canada to produce low concentrations of these undesirable substances

69. Herbicide resistant Growing a herbicide-resistant crop allows fields to be sprayed with herbicide after the crop has germinated. Glyphosate inhibits an enzyme involved in the synthesis of three amino acids: phenylalanine, tyrosine and trypot. Glyphosate is absorbed by a plant’s leaves and is transported to the growing tips. The amino acids are needed for producing essential proteins, so the plant dies The gene that was transferred into crop plants came from a strain of the bacterium Agrobacterium . Tobacco  resistant to two different herbicides: sulfonylurea and dinitroaniline

70. effects on the environment of growing a herbicide-resistant the genetically modified plant will become an agricultural weed pollen will transfer the gene to wild relatives, producing hybrid offspring that are invasive weeds herbicide-resistant weeds will evolve because so much of the same herbicide is used.

71. the evolution of resistance by the insect pests a damaging effect on other species of insects the transfer of the added gene to other species of plant. effects on the environment of growing a insect-resistant

72. Insect resistance plant – effectiveness A gene for a toxin, Bt toxin , which is lethal to insects that eat it but harmless to other animals  Bacillus thuringiensis The pollen of Bt maize (corn) expresses the gene, disperse at least 60 m by wind Experiment: caterpillars were fed milkweed leaves dusted with pollen from Bt Survival after four days of feeding: 56% Small reduction in growth of various aquatic insect larvae fed on Bt leaves. Significant effect of Caddis larvae were fed on material containing different concentrations Bt toxin.

73. Insect resistance plant – Problem Many populations of corn borers in the USA are now resistant to Bt toxin Bt resistance in corn borers happens to be a recessive allele Adult corn borers in the refuges are mostly homozygous dominant or heterozygous.

74. Golden Rice Lack of vit A was the bacground Pro-vitamin A carotenoids are in the aleurone layer of rice grains, but not in the endosperm Problem is t he aleurone layer is removed from rice when it is polished to produce white rice  rancidity Genes (carotene production) from daffodils and a common soil bacterium substituting the gene from daffodil with one from maize gave even higher quantities of carotene

75. Golden Rice

76. Genetic modification on animal A growth-hormone regulating gene  Pacific Chinook salmon Injected into a fertilised egg of an Atlantic salmon. the salmon are able to grow all year, instead of just in spring and summer characteristics of the GM salmon reduce their ability to compete with wild salmon in a natural environment.

77. Social implications The modified crop plants may become agricultural weeds or invade natural habitats. The introduced gene(s) may be transferred by pollen to wild relatives whose hybrid offspring may become more invasive. The introduced gene(s) may be transferred by pollen to unmodified plants growing on a farm with organic certification. The modified plants may be a direct hazard to humans, domestic animals or other beneficial animals, by being toxic or producing allergies. Genetically modified seeds are expensive, as is herbicide, and their cost may remove any advantage of growing a resistant crop.