MCB130

• Microbial Systems for Mutagen Detection

  
• Microbial Biosensors
• Recombinant DNA Techniques
• Applications of Microbial Genetics

  • Bacteria as test systems (mutation)
  • Microbial Biosensors (gene regulation)
  • Contributions to recombinant DNA technology
• Ames Test


  1. Determines mutagenicity of a substance

  2. Uses mutant tester strains, each with a known point mutation

  3. Salmonella typhimurium his auxotrophs

  4. TA 97, 98 ® frameshift mutant

  5. TA100 ® point mutant

  6. TA 102 ® ochre mutant

  
• Bacterial Biosensors (Microbe-based sensors - MBS)


  • Detectors of environmental pollutants
  • Living microorganisms genetically engineered to produce specific output in response to target chemicals
  • Reporter proteins are produced by the cell after specific contact or interaction with a target analyte or condition
• Reporter proteins


  • Green fluorescent protein (GFP)
  • DsRed
  • YtvA protein of Bacillus subtilis and Pseudomonas putida
  • luxCDABE of Vibrio fischeri
  • luc operon from the firefly Photinus pyrali
• Concept of a bacterial sensor-reporter cell

  • DNA parts necessary for constructing an inducible sensor-reporter circuit
  • Regulatory and reporter genes are necessary for the sensing function and system output, respectively
  • Promoter, operator(s),terminators, ribosome binding sites, etc. are DNA sequences needed for control of the gene expression

  
• Bacterial Biosensors (Microbe-based sensors - MBS)


  • Sensor function is provided by a single regulatory protein
  • Target compound is sensed by a periplasmic receiver protein that transmits the detection event via a signalling cascade to the regulatory protein
• Bioluminescence


  • Naturally bioluminescent bacteria
  • lux genes à luciferase
  • Photobacterium phosphoreum
  • Vibrio fischeri
• Bioluminescent bacteria used as biosensors

  • E. coli - for ionizing radiation
  • Acinetobacter sp - phenol, 5 - 100ppm
  • Pseudomonas fluorescens - naphthalene and salicylate in contaminated soil
  • Pseudomonas putida - benzene, toluene, xylene

  
• Applications of Bioluminescent Bacteria

  • Detection of contaminants
  • Measurement of pollutant toxicity
  • Monitoring of genetically engineered bacteria released into the environment
  • Indicators of cellular metabolic activity
  • Detection of pathogens
• Bioremediation


  The use of biological agents, such as microorganisms or their enzymes, to degrade or detoxify pollutants in a given environment and return it to its original condition
• Bioremediation methods


  • Biostimulation
  • Bioaugmentation
• Strain Improvement


  • Genetically modified to produce a specific compound or over-produce a desired product (high-yielding strains)
  • Classical methods and Recombinant DNA Technology
• Strain Improvement


  • Original Penicillium chrysogenum produced only 1.2 mg penicillin/L
  • Mutated strain produces 50g (50,000 mg) penicillin/L
• Recombinant DNA Technology (also known as genetic engineering, molecular cloning)
• Steps in making recombinant DNA

  1. Isolating the DNA

  2. Cutting the DNA

  3. Joining the DNA

  4. Amplifying the recombinant DNA

  5. Screening for the recombinant DNA
• Isolating the DNA


  • Sources of DNA: (a) plasmid or (b) chromosomal DNA
  • Cut from source or amplify polymerase chain reaction (PCR)
  • Alkaline Lysis Method for plasmid DNA
• Polymerase chain reaction (PCR)


  • In vitro amplification of DNA by synthesis of specific nucleotide sequences from a small amount of DNA
  • Generates large amounts of specific NA sequences
• PCR: Three Basic Steps

  1. Denaturation: strand separation (94oC)

  2. Annealing: primers anneal to complementary sequences (55oC)

  3. Extension: DNA synthesis (72oC)
• Restriction Enzymes


  • Recognize specific nucleotide sequences and cleave both strands of the DNA containing those sequences
  • Recognition sequences for many enzymes are the same on both strands (palindromic)

  
• Uses of Restriction Enzymes

  • To map DNA molecules physically
  • To analyze population polymorphisms
  • To rearrange DNA molecules
  • To prepare molecular probes
  • To create mutants
  • To analyze the modification status of the DNA

  
• Restriction Enzymes


  • Found in bacteria
  • Bind double strand DNA at specific sequences
  • Fragment DNA: cleave phosphodiester bonds of both DNA strands
  • Fragment ends: 5'P, 3'OH
• Over 2000 restriction enzymes from >200 prokaryotes
• Restriction Enzyme Naming


  • Enzyme name reflects bacterial strain of origin
  • e.g., EcoR V: from E. coli, strain R, 5th restriction enzyme discovered from that strain
• Host Defense Role of Restriction Enzymes

  • Cell enzymes restrict (cut) viral/other foreign DNA
  • Cell modifies & protects restriction sites in cell DNA
  • Dam (GmATC) & Dcm (CmCA/TGG) methylases
  • Methylation inhibits some restriction enzymes
• Types of Restriction Enzymes

  • Type I & III: large ATP-dependent complexes; cut and methylate DNA & bind specific sequence
  • Type II: smaller, ATP independent, only bind and cut specific sequences
• Type II Restriction Enzymes

  • Cloning tool to get specific DNA fragments
  • Recognition site usually 4-8 bp palindrome
  • Blunt/staggered cuts
  • DNA fragments with same sticky ends base pair regardless of source
• Type II Restriction Enzymes

  • Sma I (Serratia marcescens), BamH 1 (Bacillus amyloliquefaciens H) & Kpn I (Klebsiella pneumoniae)
• Restriction Enzyme Recognition Sequences

  • Variable sites (R/Y/N): Hinf I, Xho II
  • Isoschizomers - recognize same sequence, cut same way: MboI & Sau3AI (*GATC)
  • Neoschizomers - recognize same sequence, cut differently: Xma I (C*CCGGG), Sma I (CCC*GGG)
  • Isocaudomers -recognize different sequence, produce same sticky ends: BamH I & Sau3A I (*GATC)

  
• Cloning Vector


  A DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA
• Three features of a Cloning Vector

  • Sequences that permit the propagation of itself in bacteria (or in yeast for YACs)
  • Cloning site to insert foreign DNA
  • Method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA

  
• Types of Cloning Vectors

  • Plasmid
  • Phage
  • Cosmid
  • Phagemid
  • Bacterial Artificial Chromosome (BAC)
  • Yeast Artificial Chromosome (YAC)
• Plasmid


  Cloning limit of 100 to 10,000 base pairs or 0.1-10 kilobases (kb)
• Phage


  Cloning limit: 8-20 kb
• Cosmid


  An extrachromosomal circular DNA molecule that combines features of plasmids and phage; cloning limit - 35-50 kb
• Phagemids


  Plasmids that can be induced to produce phage particles containing single-stranded DNA; cloning limit up to 15 kb
• Bacterial Artificial Chromosome (BAC)


  Cloning vector
• Yeast Artificial Chromosome (YAC)


  Cloning vector