F factor Review

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F factor Review
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Mutation in Bacteria

• the ultimate source of variation in bacteria is
  spontaneous mutation,
• generally errors in DNA replication,
• mutations occur in specific genes at a rate of 1
  in 106 to 1 in 107 cells,
• adaptive mutations are quickly replicated and
  adaptive colonies predominate.

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Selection
...the process that establishes conditions in
which only the desired mutant will grow.

• 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,
• Selectable Marker gene whose expression allows
  one to identify cells in selective conditions.

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Problem
You want to create cells that are only methionine
auxitrophs.
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The Wrong Answer
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Correct Answer
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High Frequency of Recombination(Hfr)

• ...bacteria exhibiting a high frequency of
  recombination,
• the F factor is integrated into the chromosomal
  genome,
• transfer occurs at a constant rate, providing a
  means to map bacterial genes.

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Hfr and Mapping
HfrH strs (sensitive to streptomycin) thr
(able to synthesize the amino acid threonine)
azir (resistant to sodium azide) tonr
(resistant to bacteriophage T1) lac (able to
grow with lactose as sole source of carbon) gal
(able to grow with galactose as sole source of
carbon)
F- strr (resistant to streptomycin thr-
(threonine auxotroph) azis (sensitive to sodium
azide) tons (sensitive to phage T1) lac-
(unable to grow on lactose) gal- (unable to
grow on galactose)
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Hfr and Mappingkill the parents
HfrH strs (sensitive to streptomycin) thr
(able to synthesize the amino acid threonine)
F- strr (resistant to streptomycin) thr-
(threonine auxotroph)
Streptomycin kills the HfrH cells in the mating
mix.
No threonine kills the F- cells in the mating mix.
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Hfr and Mapping
HfrH azir (resistant to sodium azide) tonr
(resistant to bacteriophage T1) lac (able to
grow with lactose as sole source of carbon) gal
(able to grow with galactose as sole source of
carbon)
F- azis (sensitive to sodium azide) tons
(sensitive to phage T1) lac- (unable to grow on
lactose) gal- (unable to grow on galactose)
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Interrupting Bacterial Mating
spread on selective media
mate 9 min
blend
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Replica Platingon selective media
(streptomycin, no threonine) and
After 9 minutes, only azide resistant cells grow.
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10 Minutes
Azide, and bacteriophage resistant cells grow.
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15 Minutes
Azide, and bacteriophage resistant cells, and
lactose utilizing cells.
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18 Minutes
All recombinants grow.
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Bacterial Map Distances
units minutes
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F factor inserts in different regions of the
bacterial chromosome,
Also inserts in different orientations.
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Integration Sites (IS)Origens of Transfer
Hfr Order of transfer strain
H thr azi ton lac pur gal his gly thi 1
thr thi gly his gal pur lac ton azi
2 lac pur gal his gly thi thr azi ton
3 gal pur lac ton azi thr thi gly his
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F factor
A
a
Hfr
F-
Hfr DNA that is not incorporated in the F-
strand, and DNA that has crossed out of the F-
strand is digested.
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F factor
A transfers first.
A
A
Hfr
F-
A transfers last.
Leading Gene the first gene transferred is
determined empirically.
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Hfr Order of transfer strain
H thr azi ton lac pur gal his gly thi 1
thr thi gly his gal pur lac ton azi
2 lac pur gal his gly thi thr azi ton
3 gal pur lac ton azi thr thi gly his
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Fig. 16-9
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E. coli Map

• 0 minutes is at the threonine,
• 100 minutes is required to transfer complete
  genome,

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Typical Problem
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combine
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combine
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Refer to partial maps for map distances.
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Transformation

• heritable exchange brought about by the
  incorporation of exogenous DNA,
• usually DNA from same, or similar species.

Fig. 16.13
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Donor and Recipient
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Competence

• a transient state or condition in which a cell
  can bind and internalize exogenous DNA molecules,
• often a result of severe conditions,
• heat/cold,
• starvation, etc.

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Competent Cell
Genes are expressed that produce proteins that,
in turn, span the cell membrane.
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Exogenous DNA Binds Receptor
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Complementary Strand Degraded
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Exogenous DNA Incorporated
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Cell Divides
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Transformation and Mapping

• transformed DNA is generally 10,000 - 20,000 base
  pairs in length,
• carries more than one gene,
• When two or more genes are received from the same
  transformation event, they are said to be
  co-transformed.

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Linkage in Bacteria

• genes that are closer together, have a higher
  probability of being cotransformed,
• higher probability of being on same donor DNA,
• lower chance of crossover event between genes,
• probability of transformation by two separate
  events is low,
• linkage in bacteria refers to proximity.

Laws of Probability apply.
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Assignments

• Insights and Solutions,
• Chapter 16, Problems
• 1-12, 19