Transcript Bacteria Predominate

Week 7, 322
Monday: remove female Nasonia
Tuesday: no class
Wednesday: mt DNA
Friday: Bacteria, Conjugation
Monday, Nasonia
• Gently tap the fly pupae onto a folded piece
of paper,
• Tap adult wasps into the Morgue,
• Replace the fly pupae in the same tube,
place in incubator of choice, 18, 28 or room
temp ~24o C.
Mitochondrial DNA
-
16, 569 bp,
multiple copies per mt,
100 - 1000 mt per cell,
37 genes;
- 22 oxidative phosphorylation,
- 13 tRNA,
- 2 rRNA,
- Mitochondrial Control Region.
Mitochondrial Control Region
• control region,
– single promoter on each strand
initiates transcription,
– ori,
• D-loop,
– replication loop topography,
• hypervariable region,
– mutation rate 10x greater than
genome.
Mitochondrial Control Region
• Hair follicle DNA extraction,
• PCR,
• Sequencing (at Cold Spring
Harbor),
• Sequence analysis here at
WWU.
Link Out
Bacteria
Predominate
Bacteria Do Almost
Everything
• Metabolism;
•
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.
– Phototrophs,
– Chemotrophs,
• Biochemistry;
– ‘fix’ or synthesize a huge range of
molecules,
– break down almost anything,
– adapt to just about anything.
• Molecular Biology;
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–
–
–
–
Clone,
Gene therapy,
Eugenics,
Biotechnology,
Etc.
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.
Extra Chromosomal DNA
•
Plasmids: circular double
stranded DNA molecule that
replicates independently,
– containing one or more (nonessential) 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
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,
Growth Equals Cell Division
DNA Replication
Binary Fission
Bacterial mitosis. Why don’t bacteria do meiosis?
The (Awesome) Power of Bacterial Genetics
... is the potential for studying rare events.
Liquid Cultures,
• 109cells/microliter,
Colonies on Agar,
• 107+ cells/colony
Counting Bacteria
10-3
10-4
10-5
(Serial) Dilution is the Solution
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
• 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 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
…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,
hisleuargbio-
...can’t synthesize histidine (his+ = wt)
...can’t synthesize leucine (leu+ = wt)
...can’t synthesize arginine (his+ = wt)
...can’t synthesize biotin (bio+ = wt)
Bacterial Nomenclature
• genes not specifically referred to are considered wildtype,
– 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).
Conjugation
...temporary fusion of two single-celled organisms
for the transfer of genetic material,
…the transfer of genetic material is unidirectional.
+
-
F Cells
F Cells
(F for Fertility)
(F for Fertility)
… F+ cells donate
genetic material.
… F- cells receive genetic
material,
…there is no reciprocal transfer.
F+
F Pilus
F
-
…a filamentlike projection from
the surface of a bacterium.
F Factor
…a plasmid whose presence confers F+, or
donor ability.
-
F Pilus Attaches to F Cell
F Factor Replicates During
Binary Fission
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
...F factor
integration site,
...host (bacteria
chromosome)
integration site.
Bacterial Chromosome
Inserted F plasmid
’
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 spc thi
x
ara D(lacpro)strA thi
CSH 50:
Conjugation
Recombinant Strain:
F’lac+ proA+ proB+
ara D(lacpro)strA thi
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
...the process that establishes conditions in
which only the desired genotype will grow.
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 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.
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.
•
Extra Credit
• On another piece of paper, answer the
dilution problems on the last page of your
handout (2 pts).
Announcement
No class Monday, Tuesday 13th.
- lecture topic will be presented the preceding week.