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Bacteria Do Almost Everything. Bacteria Predominate. Metabolism; Phototrophs, Chemotrophs, Biochemistry; ‘fix’ or synthesize a huge range of molecules, break down almost anything, adapt to just about anything. Molecular Biology; Clone, Gene therapy, Eugenics, Biotechnology, Etc.

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Bacteria predominate

Bacteria Do Almost Everything

Bacteria Predominate

  • Metabolism;

    • Phototrophs,

    • Chemotrophs,

  • Biochemistry;

    • ‘fix’ or synthesize a huge range of molecules,

    • break down almost anything,

    • adapt to just about anything.

  • Molecular Biology;

    • Clone,

    • Gene therapy,

    • Eugenics,

    • Biotechnology,

    • Etc.

  • 10,000+ “Species”,

    • Mycoplasma genetalium

      • 200 nm

    • Thiomargarista namibiensis

      • 750 mm

    • soil, water, air, symbionts,

    • have adapted to aquatic and terrestrial extremes,

  • 100 grams/person,

    • 1014 bacteria.


Bacterial Chromosome

...a circular molecule of double helical DNA,

  • 4 - 5 Mb long in most species studied,

  • 1.6 mm long if brokenand stretched out.

  • Inside the cell, the circular chromosome is condensed by supercoiling and looping into a densely packed body termed the nucleoid.


  • Extra chromosomal dna
    Extra Chromosomal DNA

    • Plasmids: circular double stranded DNA molecule that replicates independently,

      • containing one or more (non-essential) genes, smaller than the bacterial chromosome,

      • may carries genes for pathogenicity,

      • may carry genes for adaptation to the environment, including drug resistance genes,

      • 1000’s of base pairs long.


    Bacterial model organism e scherichia coli e coli
    Bacterial Model OrganismEscherichia coli = E. coli

    • Enteric bacteria: inhabits intestinal tracts,

      • generally non-pathogenic,

      • grows in liquid,

      • grows in air,

    • E. coli has all the enzymes it needs for amino-acid and nucleotide biosynthesis,

      • can grow on minimal media (carbon source and inorganic salts),

    • Divides about every hour on minimal media,

      • up to 24 generations a day,





    The awesome power of bacterial genetics

    • Colonies on Agar,

      • 107+ cells/colony

    The (Awesome) Power of Bacterial Genetics

    ... is the potential for studying rare events.


    Counting bacteria
    Counting Bacteria

    10-3

    10-4

    10-5

    (Serial) Dilution is the Solution


    Model model organism
    Model Model Organism

    • Ease of cultivation,

    • Rapid Reproduction,

    • Small size,

    • Fecund (large brood size),

    • Mutants are available, stable and easy to identify?

    • Literature?

      • PubMed Listings: Eubacteria: 612,471, Archaebacteria: 9,420


    Bacteria phenotypes
    Bacteria Phenotypes

    • colony morphology,

      • large, small, shiny, dull, round or irregular,

      • resistance to bactericidal agents,

    • auxitrophs,

      • unable to synthesize raw materials from minimal media,

    • cells unable to break down complex molecules,

    • essential genes, usually studied as conditional mutants.


    Bacteria phenotypes1
    Bacteria Phenotypes

    • colony “morphology”,

      • large, small, shiny, dull, round or irregular,

      • resistance to bactericidal agents,

      • vital dyes,

    • auxitrophs,

      • unable to synthesize or use raw materials from the growth media.


    Prototroph
    Prototroph

    …a cell that is capable of growing on a defined, minimal media,

    • can synthesize all essential organic compounds,

    • usually considered the ‘wild-type’ strain.

    Auxotrophs

    …a cell that requires a substance for growth that can be synthesized by a wild-type cell,

    his- ...can’t synthesize histidine (his+ = wt)

    leu- ...can’t synthesize leucine (leu+ = wt)

    arg- ...can’t synthesize arginine (his+ = wt)

    bio- ...can’t synthesize biotin (bio+ = wt)


    Bacterial nomenclature
    Bacterial Nomenclature

    • genes not specifically referred to are considered wild-type,

      • Strain A:met bio (require methionine and biotin)

      • Strain B:thr leuthi

    • bacteriacide resistance is a gain of function,

      • Strain C:strA (can grow in the presence of strptomycin).


    Conjugation
    Conjugation

    ...temporary fusion of two single-celled organisms for the transfer of genetic material,

    …the transfer of genetic material is unidirectional.

    F+ Cells(F for Fertility)

    F- Cells(F for Fertility)

    … F+ cells donate genetic material.

    … F- cells receive genetic material,

    …there is no reciprocal transfer.


    F pilus

    F+

    F-

    F Pilus

    …a filamentlike projection from the surface of a bacterium.


    F factor
    F Factor

    …a plasmid whose presence confers F+, or donor ability.




    Properties of the f factor
    Properties of the F Factor

    • Can replicate its own DNA,

    • Carries genes required for the synthesis of pili,

    • F+ and F- cells can conjugate,

      • the F factor is copied to the F- cell, resulting in two F+ cells,

    • F+ cells do not conjugate with F+ cells,

    • F Factor sometimes integrates into the bacterial chromosome creating Hfr cells.


    Hfr cells

    ...F factor integration site,

    ...host (bacteria chromosome) integration site.

    Hfr Cells

    F factor

    Bacterial Chromosome

    Inserted F plasmid


    F cells
    F’Cells

    • an F factor from an Hfr cell excises out of the bacterial genome and returns to plasmid form,

    • often carries one or more bacterial genes along,

    • F’cells behave like an F+ cells,

      • merizygote: partially diploid for genes copied on the F’plasmid,

    • F’plasmids can be easily constructed using molecular biology techniques (i.e.vectors).


    Strain F’ genotype Chromosome Genotype

    CSH23 F’lac+ proA+proB+ D(lacpro)supE spcthi

    x

    CSH 50: araD(lacpro)strA thi

    Conjugation

    Recombinant Strain: F’lac+ proA+proB+ araD(lacpro)strA thi


    Selective media
    Selective Media

    • wild-type bacteria grow on minimal media,

    • media supplemented with selected compounds supports growth of mutant strains,

      • minimal media + leucine supports leu- cells,

      • minimal media + leucine + arginine supports leu- arg-

      • etc.

    • Selective Media: a media in which only the desired strain will grow,

      • Selective Marker: a genetic mutation that allows growth in selective medium.


    Selection
    Selection

    ...the process that establishes conditions in which only the desired genotype will grow.


    Genetic screen
    Genetic Screen

    • A system that allows the identification of rare mutations in large scale searches,

      • unlike a selection, undesired genotypes are present, the screen provides a way of “screening” them out.


    Procedure i

    Day 0: Overnight cultures of the CSH23 and CSH50 will be set up in L broth (a rich medium).

    Day 1: These cultures will be diluted and grown at 37o until the donor culture is 2-3 X 108 cell/ml. What is the quickest way to quickly determine #cells per ml? (This will be done for you.)

    Prepare a mating mixture by mixing 1.0 ml of each culture together in a small flask. Rotate at 30 rpms in a 37o shakingincubator for 60 minutes.

    At the end of the incubation…

    Do serial dilutions:

    Fill 6 tubes with 4.5 ml of sterile saline. Transfer 0.5 ml of the undiluted mating culeture to one of the tubes. This is a 10-1 dilution.

    Next make serial dilutions of 10-2, 10-3, 10-4, 10-5 & 10-6. Always change pipets and mix well between dilutions.

    Procedure I:


    Procedure ii

    Plate: 0.1 ml of a 10-2, 10-3 and 10-4 dilution onto minimal + glucose + streptomycin + thiamine.

    Plate: 0.1 ml of a 10-5 and 10-6 dilution onto a MacConkey + streptomycin plates. [A MacConkey plate is considered a rich media. It has lactose as well as other carbon sources. The phenol red dye is present to differentiate lac+ colonies (red) from lac- colonies (white).]

    Controls:

    Plate: 0.1 ml of a 10-1 dilution of donor (CSH23) cells on minimal + glucose + strep + thiamine plates. Repeat for the recipient (CSH50) cells.

    Plate: 0.1 ml of a 10-5 dilution of the recipient on a MacConkey + strep plate.

    Plate: 0.1 ml of a 10-1 dilution of donor on a MacConkey + strep plate.

    Place all plates at 37o overnight.

    Day 2: Remove the plates from the incubator the next day and count the number of white-clear colonies on the MacConkey plates (optional but easier). Store plates at 4oC. NOTE: MacConkey color reactions fade after several days or rapidly in the cold, so plates need to be scored soon after incubation.

    Procedure II:


    Extra credit

    Announcement

    • No class Monday, November 28th.

      • - lecture topic will be presented the preceding week.

    Extra Credit

    • On another piece of paper, answer the dilution problems on the last page of your handout (2 pts).


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