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Basis of course

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  1. Basis of course • Understand the technology • Understand the terminology • Gain some practical experience • The applications in biotechnology and basic biology next year • Why? • Fluidity next year (don’t need to explain terms and technology while discussing the applications) • Shows what molecular biology projects are like • Disadvantage • Technology dominated. Can make it a bit boring

  2. Lectures • Two per week, however • The Tuesday lecture may be used to discuss data from practicals • In some lecture slots the in course test will be held • Approximately formal 15 lectures

  3. Practicals • I will supervise the first two, Dianne Ford the second two • Objectives of practicals • Get used to pipetting small quantities • Reinforce lectures • Help focus on project choice for nexy year • Schedules soon

  4. BNS216 • Phases of course • Isolation of a specific DNA sequence (gene or cDNA) • Analysis of isolated DNA sequence • DNA sequencing • Manipulation of DNA sequence • PCR to introduce restriction enzyme site • PCR to change codon • PCR to detect specific DNA sequence

  5. BNS216 • Production of recombinant protein • Expression vectors • Detecting genes and transcripts • Northern hybridization • Southern hybridization • RTPCR • Manipulation of eukaryotic organisms

  6. BNS216 • Isolation of a gene • Construction of a gene library • Choice or organism • Restriction enzymes • DNA ligase • Vectors and which ones to use • Screening startegies • Prokaryotic and you look for the protein product • Eukaryotic and you look for the DNA

  7. BNS216 • Isolation of a cDNA • Purification of mRNA • DNA from mRNA • Construction cDNA library • Screening the library Analysis of DNA • DNA sequencing • Why you need to know the sequence • Automated method with fluorescent dideoxynucleotides

  8. BNS216 • Manipulation of DNA • PCR and how it relies on knowing the sequence • Introduction of restriction enzyme sites • Changing the amino acid sequence of a protein • Expression of proteins in bacteria • Expression vectors • How they enable foreign genes or cDNAs to be expressed • How expression vectors are regulated

  9. BNS216 • Detecting transcription and genes • Northern hybridization • RTPCR • Southern hybridization • Manipulation of eukaryotic multicellular organisms • Transgenic animals • Insertion of foreign genes by microinjection • Inactivating genes by homologous recombination in stem cels • Transgenic plants

  10. Assessment • Exam: • 60 % of module • Answer 3 questions from 5 • Must get >35 % to pass module • Practicals • 20 % of module • Four practicals starting on 4th February

  11. Assessment • In course tests • Four tests • Test one: Isolation of a gene or cDNA • Test two: DNA sequencing and PCR • Test three: Expression vectors and nucleic acid detection • Test four: Manipulating eukaryotic organisms

  12. BNS216 references • Difficult! • Gene Cloning: An introduction T.A. Brown OK quite simple but not in same way I teach it • Principles of Gene Manipulation: An introduction to genetic engineering R.W. Old and S.B. Primrose. Quite detailed, some of which is unnecessary • Use any standard molecular biology or genetics text book, there will a section on BNS216

  13. What is genetic engineering or recombinant DNA technology? • A suite of technologies that enable you to • Isolate and characterise genes • Produce and characterise proteins • Alter the genetic make up of an organism • New genes • Loss of existing genes

  14. Applications of genetic engineering • Basic understanding of biology • Protein structure and function • Regulation of gene expression • Importance of proteins in whole organisms (gene knockouts or null mutations)

  15. Applications of genetic engineering • Practical applications • Production of industrially important proteins • Change the properties of proteins • Modification of the phenotype of whole organisms • Diagnosis • Primary applications in medicine and agriculture • Others include chemical, paper and detergent industries

  16. Applications • More details on applications • Protein production • Pharmaceutical proteins. • Constant supply and safe • Growth hormone, insulin, Factor VIII and IX, antitrypsin

  17. Proteins • Microbial proteins • Microbes grow poorly but produce valuable enzymes • Hyperthermophiles • Anaerobes • Archaebacteria • Genetic engineering makes these proteins available to industry

  18. Enzymes used in the Food Industry • Glucose isomerase (Food industry) • Xylanases (Paper industry) • Cellulases (Energy and detergent industries) • Phytases (animal feed) • Protein engineering • Rational design • Forced protein evolution

  19. Modifying organisms • Genetically engineered foods • Herbicide resistance • Pesticide resistance • Good for consumer, farmer or biotech. Companies? • Golden rice with increased vitamin A and oil seed rape with better polyunsaturated fats • Good for consumer?

  20. Genetically engineered foods • Risks • The environment, spread of resistant weeds, alter ecological balance? • Human health. Will we get increased antibiotic resistance • Will the transgene be deleterious to human health?

  21. Change phenotype of farm animals • Convert them into bioreactors to produce pharmaceutical proteins. Why? • Change their biochemistry so • More efficient use of nutrients • Better quality end-products e.g. milk and meat • Humanising milk • Increase polyunsaturates

  22. Change phenotype of small animals • Generate animals for human disease influenced by diet • Colon cancer • nvCJD • Heart disease

  23. Gene therapy • Correct genetic defect • Not in germ line • Not transmissible

  24. Diagnosis • Diagnosis • Human genome sequenced • Identify all genes soon • Immediate diagnosis test • Hungtintons • Muscular dystrophy • Cystic fibrosis • Sickle cell anaemia • Alzeihmer • Breast cancer • Colon cancer • Heart disease • Good or bad?

  25. Diagnosis • Reduce incidence of disease • Pregnancy termination • Pre-implantation selection • Start treatment to prevent disease • Prophylactic mastectomy • Colon removal • Physiotherapy • Stress if diagnosed. Do you want to know? • Insurance and job prospects?

  26. Genetic engineering history • Pioneered by Cohen and Boyer 1972-1974 (bacterial systems) • Southern hybridization 1975 • DNA sequencing 1977-1980 • Transgenic animals 1980 • Polymerase chain reaction 1985 • Site-directed mutagenesis 1985

  27. Where do we start? • If we want to do genetic engineering how do we start? • Isolate the gene of interest • Select organism containing gene • Construct a gene library • Select members of the gene library that contain the gene of interest

  28. How do you start doing recombinant DNA technology? • Isolate the gene of interest • Lets isolate (clone) a cellulase gene • Identify organism that contains the gene • Rumen • compost • Soil • Leaf litter • Decaying wood

  29. Isolating a cellulase gene Isolate chromosomal DNA Fragment DNA

  30. Mix and ligate Vector E. coli Transform Gene library

  31. Properties of vector DNA • Replicates in bacterium • Foreign DNA inserted will be stable • Normally extra-chromosomal • Easy to select bacterium containing vector (confers antibiotic resistance) • Vectors • Plasmid (extra chromosomal circular DNA) • Bacteriophage • Cosmids • Artificial chromosomes

  32. Gene library Screen library for appropriate gene (cellulase gene) Isolate plasmid

  33. Isolating a cellulase gene Isolate chromosomal DNA Fragment DNA

  34. Restriction endonucleases • Enzymes that cut DNA at specific sequences • Discovered in the early 1950s • Agent that enables bacteria to be immune to bacteriophage • Host-controlled restriction • Mainly found in bacteria • Over 1200 characterised

  35. Restriction endonucleases • Three types only Type II important in genetic engineering as they cut the sequence they recognise • Target sequences generally palindromic • Recognise 4, 6 or 8 nucleotides

  36. Restriction endonucleases EcoRI GAATTC CTTAAG G AATTC CTTAA G Sticky ends PstI CTGCAG GACGTC CTGCA G G ACGTC SmaI CCC GGG GGG CCC CCCGGG GGGCCC Blunt ends

  37. Restriction endonucleases • Named after organism • e.g. EcoRI = Escherichia (E) coli (co) strain R (R). I refers to Ist enzyme isolated from organism • Why doesn’t a bacterial restriction endonuclease digest its own DNA? • The bacterium produces a DNA methylase that recognises same sequence as restriction endonuclease • Methylates target DNA sequence which makes it resistant to endonuclease cleavage

  38. Restriction enzymes and DNA methylase Foreign DNA Host DNA GAATTC CTTAAG GAATTC CTTAAG EcoRI DNA methylase CH3 G AATTC CTTAA G GAATTC CTTAAG CH3 EcoRI CH3 GAATTC CTTAAG CH3

  39. Mix and ligate Vector E. coli Transform Gene library

  40. Forming hybrid or recombinant DNA molecules using restriction enzymes and DNA ligase GAATTC CTTAAG GAATTC CTTAAG Digest with EcoRI G AATTC CTTAA G G AATTC CTTAA G Mix DNA GAATTC CTTAAG GAATTC CTTAAG GAATTC CTTAAG DNA ligase DNA ligase GAATTC CTTAAG GAATTC CTTAAG GAATTC CTTAAG Recombinant or hybrid DNA

  41. Inserting chromosomal DNA into a vector Chromosome GAATTC CTTAAG GAATTC CTTAAG Vector GAATTC CTTAAG Cut with EcoRI and add DNA ligase Recombinant vector GAATTC CTTAAG GAATTC CTTAAG

  42. More details on each stage • Chromosomal DNA is only partially cut because? • Don’t know if the restriction enzyme cuts in the gene • Plasmid vector is designed to enable selection for recombinant plasmid • pUC or pBluescript-based plasmid vectors • Contains two selection genes ampicillin (antibiotic) and LacZ; codes for -galactosidase

  43. HindIII EcoRI BamHI pUC18 Cells containing pBluescript are ampicillin resistance and blue on X-Gal Origin of replication LacZ’ encodes -galactosidase Ampr confers ampicillin resistance

  44. Bromo-chloro-indoyl--galactopyranosidase or X-Gal (Clear) -galactosidase Bromo-chloro-indoyl (Deep blue insoluble) + galactose

  45. Inserting chromosomal DNA into a vector Chromosome GAATTC CTTAAG GAATTC CTTAAG Vector GAATTC CTTAAG Cut with EcoRI and add DNA ligase Recombinant vector GAATTC CTTAAG GAATTC CTTAAG Ampicillin resistant; -galactosidase negative (White on X-Gal) LacZ gene codes for -galactosidase Ampicillin resistance gene

  46. Wild type vector GAATTC CTTAAG Ampicillin resistant; -galactosidase active (Blue on X-Gal) LacZ gene codes for -galactosidase Ampicillin resistance gene

  47. E. coli sensitive to ampicillin Ampicillin resistant; -galactosidase negative (White on X-Gal) Ampicillin resistant; -galactosidase active (Blue on X-Gal)

  48. Bacteria from ligation platedon ampicillin and X-Gal Contains wild type plasmd Contains recombinant plasmd

  49. Gene library • Collection of microbes (e.g. Escherichia coli) each one containing a recombinant vector • Each recombinant vector contains a random region of the target chromosome • The number of microbes in the library is large • Thus any gene in the target organism’s genome is present in at least one member of the gene library

  50. Mix and ligate Vector E. coli Transform Gene library