Transcript Bacteria Predominate - Western Washington University

Arabidopsis Experiments:
I.
Forward Genetic Screen (Ethylene Insensitive Mutants):
•
requires thinking.
II. Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants):
•
requires scoring F2 and thinking.
What Next?
…experiment I
dominant
Thought Experiments…
 Backcross to wild-type,
 what might the F1 and F2 tell us?
 Complementation tests?
 Given etr1, etr2, ers1, ers2, ein4,
ctr1, ein2, ein3, eil1 and erf1
homozygous plants, and wt
plants; devise a plan to describe
the genetic nature of the 12 long
hypocotyl mutants you found.
recessive
What Next?
…experiment II
L t T
homozygote
wt
5’
5’
3’
3’
L t T
5’
3’
5’
3’
heterozygote
homozygote
mutant
5’
3’
5’
3’
L t T
Genetic Analysis
F2 Segregation (Friday)
…what next?
T
t
T
TT
Tt
t
Tt
tt
1:2:
1
Not Lethal
T
t
T
TT
Tt
t
Tt
tt
1 wt : 2 het
Lethal
How would you confirm / extend F2 results?
T
t
T
TT
Tt
t
Tt
tt
1 wt : 1 het
Gametophyte
Lethal
Genetic Selection
...the process that establishes conditions in
which only the desired genotype will grow.
Selective Media: what might this be?
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.
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
Extra Credit: On another piece of paper, answer the dilution problems
on the last page of your handout (2 pts), due Thursday, 13th.
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).
Transfer of lac+pro+ from a F' to an F- strain.
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•
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Strain
CSH23
CSH50
Sex
F’ lac+ proA+ proB+
F-
Genotype
D(lacpro) supE spc thi
ara D(lacpro) strA thi
strA: confers resistance to streptomycin
spc: confers resistance to spectinomycin
D indicates a deletion of the genes in parentheses
lac: cannot utilize lactose as a carbon source
pro: indicates a requirement for proline
thi; indicates a requirement for thiamine
supE: suppresses nonsense mutations
ara: cannot utilize arabinose as a carbon source.
•
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
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.
•
What’s Growing?
…mate in rich, transfer to…
•
Plate: minimal + glucose +
streptomycin + thiamine:
– CSH23
yes / no
– CSH50
yes / no
– exconjugate yes / no
•
Plate: MacConkey (rich) +
streptomycin plates:
– CSH23
yes / no
– CSH50
yes / no
– exconjugate yes / no
CSH23
F’lac+ proA+ proB+
D(lacpro)supE spc thi
x
ara D(lacpro)strA thi
CSH 50:

F’lac+ proA+ proB+
ara D(lacpro)strA thi
Extra Credit
• On another piece of paper, answer the
dilution problems on the last page of your
handout (2 pts), due with your abstract on
Weds.