molecular microbiology

• •Cloning:Replication of specific DNA fragment in a relevant host organism
• Plasmid cloning:

  • PCR amplification of gene using specific primers: Denaturing, annealing, elongation
  • Engineer restriction sites that correspond to multiple cloning site (MCS)
  • Ligate (DNA Ligase)
  • Transform host (e.g. E. coli)
  • Selection (antibiotic resistant?)
  • Promoter: Constitutive/inducible
• plasmid cloning:

  • restriction enzymes cut dna forming sticky ends
  • dna ligase rejoins cut dna with the plasmid at the multiple cloning site.
  • insterted back into host for replication
• constitutive= host cell constantly produces molecules

  inducible= by changing the environment you can decide whether to turn production on or off
• The lac promoter:

  • lac repressor binds to promotor region, prevents transcription in the presence of glucose
  • in the presence of IPTG, lac repressor is removed and rna polymerase binds initiating transcription.
• chemical transformation:

  • wash bacteria in cold calcium chloride
  • prepares membrane and cell wall to take up substances
  • heat shock
  • take up plasmid from environment
• electroporation:

  • bacteria washed with cold glycerol
  • plasmid and host cell are together
  • electric shock passed through plasmid taken up
  • test for antibiotic resistance
• •Chemostatic regulation of growth environment (fermentation)

  • temperature, pH, nutrient, pO2
• Insulin:

  • processed product:
  • first it is in linear form (preproinsulin)-> folds into insulin
  • Two recombinant plasmids carrying artificial genes for A/B chain which code for the active part of insulin
  • Controlled by lac promoter
• Hepatitis B vaccine:

  • Can’t be grown in culture
  • Vaccines initially made from viruses extracted from the blood of carriers
  • Subunit vaccine:
  • HBsAg – envelope protein
  • Major antigenic determinant of the virus
  • Production in E. coli – not effective
  • Production in yeast – adopts natural conformation
  • Cheap, safe, overcomes culture issue
• Cloning into eukaryotes:

  • E.g. Saccharomyces cerevisiae or Pichia pastoris:
  • Can process proteins, Glycosylation, Sugars added to asparagine residue, Carried out in ER, Further modification in Golgi= Affect protein folding, activity, immune recognition and half-life
  • Yeast can grow in culture easily
  • Good genetics – different plasmids
  • Promoters differ
  • E.g. GAL1 galactokinase
• 1. Yeast (Saccharomyces cerevisiae)= model organisms, ease of cultivation in culture, well-established genetic tools, carry out genetic manipulations using different plasmids.
  2. Different Plasmids and Promoters: Yeast cells can be transformed with various plasmids carrying different selectable markers and regulatory elements, including promoters. For example, the GAL1 promoter drives the expression of genes in response to galactose induction, providing a tool for controlled gene expression in yeast.
• microorganisms can be used to make biological warfare.
• Anthrax toxin;

  • Produced by Gram positive Bacillus anthracis
  • Can produce highly resistant endospores
  • Can be aerosolised
  • Spores remain viable for years
  • 3 forms: cutaneous, inhalation and gastrointestinal
  • >80% mortality when untreated
  • Death from septicaemia, haemorrhagic meningitis
  • Anthrax toxins:
  • PA (protective antigen) - B moiety (vaccine)
  • EF(oedema factor), LF (lethal factor)= A moiety
• Botulinum toxin:

  • Spore forming Gram positive obligate anaerobe
  • Disease results from absorption of toxin into blood stream
  • Causes flaccid paralysis (prevents ACH release)
• During the process of recombinant protein production, the enzyme Ligase performs what function?


  cleavage of double stranded dna
• During the process of recombinant protein production, the enzyme DNA polymerase performs what function?


  Duplication of double stranded dna
• Cleavage sites of restriction endonucleases are typically found in what region of a cloning plasmid?


  Multiple cloning site
• Restriction endonucleases can cleave double stranded DNA to produce blunt ended DNA or sticky ends
• The Lac promoter controls transcription in response to lactose, glucose, IPTG
• Selective


  Allows for the growth of a specific organism
• Chromogenic


  Colourful plates for specific organisms
• Types of bacterial culture media


  • Liquid culture
  • Solid culture
• Liquid culture - Growth media


  Allows for the growth of most organisms
• Liquid culture - Differential media


  Distinguishes between different organisms
• Solid culture - Non-selective


  Allows for the growth of most organisms
• Solid culture - Differential


  Distinguishes between different organisms
• Growth liquid media - Luria broth

  1. Tryptone 10 g

  2. Yeast Extract 5 g

  3. NaCl 10 g
• Growth liquid media - Cooked meat medium


  1. Cooked Meat 250.0g

  2. Dextrose 5.0 g

  3. NaCl 5.0 g

  4. Yeast Extract 5.0 g

  5. Iron Filings 10.0 g

  6. Hemin 10.0 ml

  7. Vitamin K 10.0 ml
• When the media is cloudy it indicates bacterial growth
• Differential liquid media - Phenol red lactose fermentation broth


  1. Peptone 10.g

  2. Beef Extract 1g

  3. Sodium Chloride 5g

  4. Phenol Red 0.018g- indicator

  5. Lactose 5g- enzyme lactase
• Differential liquid media - Organisms


  • E.coli
  • Pseudomonas
• Agar


  Cannot be degraded by most bacteria, originally used as food thickener
• Angelina Fanny Hesse and Walther and Fanny Hesse joined Robert Koch's laboratory to isolate bacteria from the air

  1880s
• Potato based medium not successful, beef broth solidified with gelatin turned into a cloudy liquid overnight as bacterial enzymes broke down the gelatin as they grew
• Non-selective solid media - Nutrient agar


  1. 5g peptone

  2. 3g beef extract/yeast extract

  3. 15g agar

  4. 8g NaCl

  5. 1000ml Distilled water
• Enriched agar - Blood agar

  1. 5 - 10% sheep or horse blood

  2. 0.3 % beef / yeast extract

  3. 0.5 % peptone

  4. 1.5 % agar

  5. 0.5% NaCl
• Haemolysis on blood agar


  Alpha haemolytic = The reduction of the red blood cell haemoglobin to methaemoglobin

  Beta haemolytic = Complete or true lysis of red blood cells

  Gamma haemolytic = Lack of haemolysis
• Some bacteria produce hemolysins that lyse red blood cells and degrade hemoglobin
• Selective Culture Media


  Elective agents - Encourage growth of desired organism

  Selective agent - Desired organisms are resistant

  Differential agents - Discriminate desired organism from others