1. Microbial Genetics and biotechnology

2. Define Terms  Genetics  Genome / Genomics  Chromosomes  Gene  Genotype  Phenotype  Recombination

3. Control of Gene Expression

4. DNA Structure  Double stranded  Nucleotide Nitrogen Bases Sugar Phosphate  Base Pairs Hydrogen Bonds  A-T  C-G Alpha helix  5’ – phosphate group  3’ – hydroxyl group

5. RNA Structure  Single strand  Nucleotide Nitrogen base Sugar Phosphate  Base Pairs A-U C-G  Three types mRNA rRNA tRNA

6. Prokaryotic Chromosomes  Location Nucleoid region No membrane  Number Most have 1 Some species have 2, the second linear  Appearance Circular Ds Loops and coils

7. E. coli genome / chromosome

8. DNA Replication  Semiconservative Replication fork  Single origin  Bidirectional 2 Leading strands 2 Lagging strands Enzymes  Helicases  DNA polymerases 5’ to 3’ I for leading strand II digest RNA primer III for lagging strand  DNA ligase  DNA gyrase Hydrogen bonds broken and reformed Methylation of adenine bases  Initiation sites for replication  Turn on or Turn off gene expression  Protect against viral infections  DNA repair Polymerase I & II

9. DNA Replication Enzymes  DNA Helicase  DNA Primase  DNA Polymerase  DNA Gyrase  Topoisomerases  DNA Ligase

10. Leading/Lagging Strands

11. DNA Replication Overview

12. Binary Fission

13.  Asexual reproduction  DNA replicated  FTs proteins Divisome apparatus  Peptidoglycan  Plasma membrane  Double numbers

14. Plasmids

15.  2% of genetic information (5-100 genes)  ds, circular extra chromosomal DNA  Independent replication  Cellular Traits F-Fertility R-Resistance : inactivate AB, toxins, heavy metals Dissimilation: catabolism of unusual substances Bacteriocins Virulence : enzymes, toxins, attachment

16. Rolling Method for DNA replication and F-Plasmid  Rolling Method One strand remains in loop Second strand breaks away and rolls of loop Both strands serve as templates for daughter strand Occurs during conjugation

17. Plasmid Integration (Episome)

19. Transcription  DNA  RNA  mRNA  rRNA  tRNA  Initiation Sigma factor on RNA polymerase  binds to promoter sequence on DNA  Will be release after 10 nucleotides RNA polymerase  unzips, unwinds DNA  Lacks proof reading ability  Elongation 5’ to 3’, slower  Ribonucleotide sequences Base pairs : A-U [instead of Thymine] C-G  Termination Self  Terminator sequence  G-C rich area Protein-dependant  Terminator protein  Separates DNA and RNA polymerase

20. Sigma Factors for RNA polymerase Where RNA polymerase binds to DNA

21. Prokaryotic RNA  Transcription = RNA  Polypeptides  RNA mRNA  Code for several polypeptides along strand  Each code has codons: Start and Stop tRNA  Acceptor stem  Anticodon  Wobble rRNA  70S Ribosomes 50S: 23S + 5S rRNA and 33 proteins 30S: 16S rRNA and 21 proteins  Binding Sites on Ribosomes A: accepts tRNA with AA P: holds tRNA for base pairing anticodon to mRNA codon for polypeptide E: release [Exit] for tRNA

22. Translation Steps  Initiation 30S tRNA @ P site 50S GTP used  Elongation New tRNA @ A site Ribozyme in 50S forms peptide bond GTP used  Termination Release factor proteins Stop codon on mRNA

23. Importance of rRNA structures

25. Regulation of Gene Expression  Constitutive Not regulated Always “on” at fixed rate  Transcription  Translation 60-80% Polypeptides need in large amounts  Regulated Only when needed Control synthesis of genes for enzymes  Induction  Repression Control enzyme activity: feedback  Noncompetitive inhibition  Competitive inhibition

26. Enzyme Reguation

28. Operon Parts  Operator Controls access On/off  Promoter RNA polymerase binds All or none  Regulator Genes at distant site control transcription Repressor binds to operator to block  Structural genes Code for enzymes

29. Operon regions on DNA

30. Operation of Operon Gene expression

31. Operator Always “on” unless switched off by repressor

32. Promoter Region

33. Regulation of Operator Genes  Negative Control Repressor On/Off Interacts with operon Types  Inducible  Repressible  Positive Control Activator protein Determines rate Directly interacts with genome Facilitates transcription

34. General Regulatory Control animation

35. “Negative” Genetic Control of Enzyme synthesis and formation  Operon Model Operator (O) Promoter (P) Structural genes  Regulatory genes Makes repressor  Active binds to Operator  Inactive unable to bind to Operator  Types Inducible Operons  Repressor Active  Operon Off  Inducer needed  Catabolic Pathways Repressible Operons  Repressor inactive  Operon On  Corepressor needed  Anabolic pathways

36. Repressor Proteins  Regulatory  Control Gene expression  Binds to operator  Cues from metabolites Active form = blocks Inactive form = allows transcription

37. Active Repressor Proteins

38. Inactive Repressor Proteins

39. Inducible Operon  Repressor Active Bound to operon  Operon off  Need Inducer to inactivate repressor  Inducer metabolite that can bind to repressor Inactivates repressor  Operon “induced” on

40. Lac Operon: Inducible

41. Use of Lactose

42. Repressible Operon  Repressor inactive  Operon on  Need co-repressor Metabolite Binds to repressor Activates repressor

43. TRP Operon: Repressible

44. “Negative” Gene Control

45. Which Regulatory Gene Operon is this?

46. The “other” one  Positive Control of Gene Expression  Catabolite Activator Protein (CAP) Binds to promoter region Enhance affinity for RNA polymerase Stimulate gene expression

47. CAP (Catabolite Activator Protein)  cAMP binds and activates CAP (crp)  cAMP-CAP bind to promoter  Increase RNA polymerase affinity  Allow efficient transcription  Determines rate No cAMP, no CAP binding, rate slows

49. Mutations  Define  Types Silent Point  Mis-sense  Non-sense  Sense [aka silent] Substitution  Transition: purine for purine  Transversion: purine for pyrimadine Frameshift  Insertions  Deletions  Causes Spontaneous Induced  Chemical  Physical Conditional Adaptive Transposons Thymine dimer (radiation) Inversion (transposons)

51. Repair of Mutations

52. Transposons (Transposable elements)  DNA fragments within chromosomal DNA  Gene producing enzymes for insertion  Types Simple  Insertion only Complex  Additional genes Jumping genes Move  Within one chromosome  From one chromosome to another

53. Transposon Jumping Patterns  Conservative Simple Not replicated Move pre-existing  Replicative Complex Copies Original in tact New site with new genes (jump)

54. Transposon copies

55. Genetic Transfer & Recombination  Vertical Parent to offspring  Horizontal Lateral transfer to same generation Donor to recipient DNA transfer Plasmid transfer Types  Transformation  Transduction  Conjugation

56. Transformation  Occurance 1% Random Naturally in certain species  Haemophilus  Neisseria  Pseudomonas  Streptococcus  Staphylococcus  Griffith experiment  Genetic transfer Environmental surroundings Naked DNA assimilated Competent cells  Cell wall  Plasma membrane Bacterial lysis  DNA  Plasmids

57. Griffith Experiments

58. Transduction  Transfer of bacterial genes via viruses Donor to recipient Virus: Bacteriophages  Types Generalized Specialized  Replication Cycle Lytic Lysogenic

59. Transduction

60. Generalized Lytic Cycle  Random pieces of host cell DNA (any genes)  Packaged with phage during lytic cycle  Donor DNA combines with recipient

61. Lytic Cycle Summary

62. Specialized Transduction Cycle  Only certain specific bacterial genes are transferred  Example: Toxins Corynebacterium  Diphtheria toxin Streptococcus pyogenes  Erythrogenic toxin E. coli  Shiga-like toxin

63. Random genes transferred Specific Genes transferred

64. Lysogenic Cycle Summary

65. Conjugation Sex

66. Conjugation  Define  Bacteria Gram Neg : F.pilus Gram Pos: sticky surface molecules  Types F+ [plasmid] R [plasmid] Hfr [DNA]

67. Conjugative Plasmid

68. Plasmid F-factor  F = fertility F+ = male F- = female  ~25 genes  Sex Pilus  Replicates in synchrony with host DNA  Rolling method  DNA replicates from parent  If integrated with host DNA becomes Hfr

69. Hfr Interrupted Stages

70. Other Plasmids  R factors Resistance to AB ~10 genes Different bacterial species share  Bacteriocin factors Specific protein toxins Kill other cells of same or similar species  Virulence Pathogenicity  Structures  Enzymes  Toxins

71. Conjugation: R Plasmid transfer

72. Genetic Recombination  General Homologous chromosomes Any location DNA breakage and repair  Site Specific Non-homologous Viral genomes in bacterial chromosomes  Replicative  Health and Food industries

73. Recombinant DNA

74. Genetic Engineering  Use Plasmids Recombinant DNA  Applications Therapeutic  Hormones  Enzymes  Vaccines  Gene therapy Agricultural Scientific  Southern Blot  ELISA tests

76. Biotechnology

77. Questions?