Bacteria Predominate 10,000+ “Species”, –Mycoplasma genetalium 200 nm – Thiomargarista...
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Transcript of Bacteria Predominate 10,000+ “Species”, –Mycoplasma genetalium 200 nm – Thiomargarista...
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Bacteria Predominate
• 10,000+ “Species”,
– Mycoplasma genetalium • 200 nm
– Thiomargarista namibiensis• 750 m
– soil, water, air, symbionts,– have adapted to aquatic and
terrestrial extremes,
• 100 grams/person,
– 1014 bacteria.
• 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.
Bacteria Do Almost Everything
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Bacterial Chromosome
...a circular molecule of double helical DNA,
– 4 - 5 Mb long in most species studied,
– 1.6 mm long if broken and stretched out.
• Inside the cell, the circular chromosome is condensed by supercoiling and looping into a densely packed body termed the nucleoid.
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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.
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Bacterial Model Organism Escherichia 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,
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Growth Equals Cell Division
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DNA Replication
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Binary Fission
Bacterial mitosis. Why don’t bacteria do meiosis?
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The (Awesome) Power of Bacterial Genetics
... is the potential for studying rare events.
Liquid Cultures,
• 109cells/microliter,
Colonies on Agar,• 107+ cells/colony
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Counting Bacteria
(Serial) Dilution is the Solution
10-3 10-510-4
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Model Model Organism
• Ease of cultivation,Ease of cultivation,
• Rapid Reproduction,Rapid Reproduction,
• Small size,Small size,
• Fecund (large brood size),Fecund (large brood size),
• Mutants are available, stable and easy to identify?Mutants are available, stable and easy to identify?
• Literature?Literature?
• PubMed Listings: Eubacteria: 612,471, Archaebacteria: PubMed Listings: Eubacteria: 612,471, Archaebacteria:
9,4209,420
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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.
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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.
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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)
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Bacterial Nomenclature
• genes not specifically referred to are considered wild-type,
– Strain A: met bio (require methionine and biotin)
– Strain B: thr leu thi
• bacteriacide resistance is a gain of function,
– Strain C: strA (can grow in the presence of strptomycin).
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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 donate genetic material.
… F- cells receive genetic material,
…there is no reciprocal transfer.
F- Cells(F for Fertility)
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F Pilus
…a filamentlike projection from the surface of a bacterium.
F+
F-
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F Factor…a plasmid whose presence confers F+, or
donor ability.
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F Pilus Attaches to F- Cell
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F Factor Replicates During Binary Fission
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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.
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Hfr Cells
F factor
Bacterial Chromosome
Inserted F plasmid
...F factor integration site,
...host (bacteria chromosome) integration site.
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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).
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Strain F’ genotype Chromosome Genotype
CSH23 F’lac+ proA+ proB+ (lacpro)supE spc thi
x
CSH 50: ara (lacpro)strA thi
Conjugation
Recombinant Strain: F’lac+ proA+ proB+ ara (lacpro)strA thi
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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.
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Selection
...the process that establishes conditions in which only the desired genotype will grow.
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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.
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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 shaking incubator 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.
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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.
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Extra Credit
• On another piece of paper, answer the dilution problems on the last page of your handout (2 pts).
Announcement
No class Monday, November 28th.
- lecture topic will be presented the preceding week.