Arabidopsis Experiments:

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Arabidopsis Experiments

• Forward Genetic Screen (Ethylene Insensitive
  Mutants)
• requires thinking.
• II. Reverse Genetic Screen / PCR Genotyping (H-
  ATPase Mutants)
• requires scoring F2 and thinking.

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What Next?experiment I
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.

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What Next?experiment II
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Genetic AnalysisF2 Segregation (Friday)what
next?
How would you confirm / extend F2 results?
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(No Transcript)
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Genetic Selection

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

Selective Media what might this be?
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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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The (Awesome) Power of Bacterial Genetics

• ... is the potential for studying rare events.

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Counting Bacteria
10-3
10-5
10-4
(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.
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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-
...cant synthesize histidine (his wt) leu-
...cant synthesize leucine (leu wt) arg-
...cant synthesize arginine (his wt) bio-
...cant 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(F for Fertility)
F cells donate genetic material.
F- cells receive genetic material, there is
no reciprocal transfer.
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F Pilus
a filamentlike projection from the surface of a
bacterium.
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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
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FCells

• 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,
• Fcells behave like an F cells,
• merizygote partially diploid for genes copied on
  the Fplasmid,
• Fplasmids can be easily constructed using
  molecular biology techniques (i.e.vectors).

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Transfer of lacpro from a F' to an F- strain.

• Strain Sex Genotype
• CSH23 F lac proA proB
  D(lacpro) supE spc thi
• CSH50 F- ara D(lacpro) strA thi
• strA confers resistance to streptomycin
• spc confers resistance to spectinomycin
• 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.

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• Strain F genotype Chromosome Genotype
• CSH23 Flac proA proB D(lacpro)supE
  spc thi
• x
• CSH 50 ara D(lacpro)strA thi

Conjugation
Recombinant Strain Flac proA proB
ara D(lacpro)strA thi
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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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Whats Growing?mate in rich, transfer to

• CSH23 Flac proA proB D(lacpro)supE
  spc thi
• x
• CSH 50 ara
  D(lacpro)strA thi
• ?
• Flac proA proB ara
  D(lacpro)strA thi

• 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

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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.