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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.
slide3

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

Liquid Cultures,

    • 109cells/microliter,
  • 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).
slide24
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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