Bacterial Genetics, Biotechnology, And Recombinant DNA PDF

BACTERIAL GENETICS BIOL 220 MICROBIOLOGY Thursday / September 19, 2024  COURSE OBJECTIVES 1. Define genotype, phenotype, mutation, vertical and horizontal gene transfer, and wild type. 2. Discuss the types and causes of spontaneous and induced mutations...

BACTERIAL GENETICS BIOL 220 MICROBIOLOGY Thursday / September 19, 2024  COURSE OBJECTIVES 1. Define genotype, phenotype, mutation, vertical and horizontal gene transfer, and wild type. 2. Discuss the types and causes of spontaneous and induced mutations 3. Describe repair of errors in nucleotide incorporation, modified nucleobases, and thymine dimers, and the mechanism of SOS repair 4. Define auxotroph and prototroph and explain how The Ames Test is used to screen possible carcinogen 5. Discuss horizontal gene transfer; DNA-mediated transformation, 2 transduction and conjugation BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA 9/17/2024  GENETIC CHANGE IN BACTERIA  Genotype (the sequence of nucleotides) and phenotype (observable characteristics)  Two mechanisms  Mutation: change in the existing nucleotide sequence, results in vertical gene transfer  Horizontal gene transfer: movement of DNA from one organism to another  Wild type: a typical phenotype of strains isolated from nature 3 9/17/2024  SPONTANEOUS MUTATION (1)  Caused by normal processes, passed to progeny, occurs randomly (rate: 10-4 to 10-12)  Occasionally change back to original state (reversion)  Base substitution  The most common  Point mutation: change in one base pair  Three possible outcomes: silent, missense, or nonsense mutation 4 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  5 9/17/2024  SPONTANEOUS MUTATION (2)  Deletion or addition of nucleotides  Impact depends on location and number of nucleotides  Frameshift mutation  Adding or subtracting one or two nucleotide pairs  Often results in premature STOP codon: shortened, nonfunctional protein (“knockout mutation”) 6 9/17/2024  SPONTANEOUS MUTATION (3)  Transposons (or “jumping genes”)  Can move from one location to another : transposition  Gene inactivated  Most transposons have transcriptional terminators: stops downstream gene expression 7 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  INDUCED MUTATIONS (1)  From outside influence 8 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  INDUCED MUTATIONS (2)  Chemical mutagens: 3 groups  Chemical that modify nucleobases: increases chance of incorrect nucleotide incorporation, eg. alkylating agents (Nitrosoguanidine) 9 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  INDUCED MUTATIONS (2)  Base analogs: resembles nucleotide, eg. 5-bromouracil resembles thymine, 2-amino purine resembles adenine  Intercalating agents: increases chances of frameshift mutations, eg. ethidium bromide and chloroquine 10 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  INDUCED MUTATIONS (3)  Radiation  Ultraviolet light: forms thymine dimers, distorts molecule  X-ray: cause single- and double-strand breaks and changes in nucleobases 11 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  REPAIR OF DAMAGED DNA (1)  Repair of errors in nucleotide incorporation  Proofreading by DNA polymerase: verifies accuracy, efficient but not flawless  Mismatch repair: enzyme cuts sugar-phosphate backbone, DNA polymerase and DNA ligase make repairs  Repair of thymine dimers  Photoreactivation: using light and enzymes  Excision repair 12 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  REPAIR OF DAMAGED DNA (2)  Repair of modified nucleobases  SOS repair (“SOS mutagenesis”):  Last-ditch effort to repair extensively- damaged DNA  DNA and RNA polymerases stall at unrepaired sites  Several dozen genes in SOS system activated: DNA polymerase that synthesizes even in extensively damaged regions  Has no proofreading ability, so errors made 13 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  AMES TEST  Auxotroph: a mutant that requires a growth factor  Prototroph: does not require a growth factor  A method to test for possible carcinogen  Most carcinogens are mutagens (increase frequency of spontaneous reversion)  Measures effect of chemical on reversion rate of histidine- auxotroph Salmonella 14 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  HORIZONTAL GENE TRANSFER  Transformation: uptake of DNA by bacteria  Transduction: transfer of DNA via a phage or bacterial viruses  Conjugation: DNA transfer between bacterial cells 15 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA BIOTECHNOLOGY AND RECOMBINANT DNA BIOL 220 MICROBIOLOGY Thursday / September 19, 2024  COURSE OBJECTIVES 1. Describe the role of restriction enzymes and gel electrophoresis in biotechnology 2. Give some applications of genetically engineered bacteria and plants 3. Discuss some of the concerns regarding genetic engineering 4. Describe polymerase chain reaction (PCR) 5. Compare and contrast different probe technologies: blotting, FISH and DNA microarray 17 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  RESTRICTION ENZYMES o Recognize 4 to 6 base-pair nucleotide sequence and cut each strand of DNA, generating restriction fragments o Cohesive ends (sticky ends) that are complementary can anneal o Allows creation of recombinant DNA molecules 18 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  GEL ELECTROPHORESIS  Separates DNA based on size  Electrical current: DNA migrates toward the (+) electrode 19 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  GENETICALLY ENGINEERED BACTERIA (1)  DNA cloning 20 9/17/2024  GENETICALLY ENGINEERED BACTERIA (2)  Protein production: safer, more economical  Hormone: insulin  Vaccine: HepB and cervical cancer  Cheese manufacturing: chymosin (rennin)  “Synthetic bacteria”: may someday produce wide range of commercially valuable substances  DNA production: research purposes  “Shotgun cloning”: cloning of random DNA samples from environment 21  Studying gene functions and regulation 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  GENETICALLY ENGINEERED EUKARYOTES  Transgenic: engineered plants and animals  Corn, cotton and potatoes engineered to produce Bt toxin (biological insecticides)  Soybean, cotton, corn engineered to resist biodegradable herbicide glyphosate (“Roundup”)  Plants with improved nutritional value, eg. β-carotene and iron in rice  Edible vaccines 22 9/17/2024 transgenic rice wild type  CONCERNS REGARDING GENETIC ENGINEERING  New technologies should be tested for safety and effectiveness  Recombinant DNA Advisory Committee (RAC) formed by NIH  Numerous advances may be used for malicious purposes  Concerns over sequencing human DNA: ethical issues  The Genetic Information Nondiscrimination Act (GINA)  Genetically modified (GM) organisms: logical and non logical concerns  GM foods “contain DNA”  Concerns over possible allergens: leads to strict guidelines  Unintended effects on environment: pollen from Bt plants and 23 monarch butterflies, herbicide-resistant genes may transfer to weeds 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  POLYMERASE CHAIN REACTION (PCR)  Allows amplification to millions of copies of DNA in a matter of hours  Thermal cycler  Detection of organisms without culturing  Requires template DNA, heat-stable polymerase (Taq), primers, deoxynucleotides  Three-step amplification cycle  DNA denaturation: ~95°C  Primer annealing: 45 - 54°C  DNA synthesis: ~70°C 24 9/17/2024  PROBE TECHNOLOGIES (1)  DNA probes: locate specific nucleotide sequence  Annealing to its complementary sequence (“hybridization”)  Labeled with a marker  Colony blotting  Detection of colonies that have specific sequence of DNA 25 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA  PROBE TECHNOLOGIES (2)  Fluorescence in situ Hybridization (FISH)  Cells fixed to a microscope slide, probe hybridizes to nucleotide sequences, visualized via fluorescence microscopy  Probe may go after specific group or population  DNA microarrays (“gene chips” or “biochips”)  Studies gene expression: mRNA is isolated, reverse transcription to produce cDNA 26 9/17/2024 BIOL220 Chapter 8-9 Bacterial Genetics_Biotechnology and Recombinant DNA