Lecture 9 Objectives: Students should be able to:

Design a screen for isolating antibiotic resistance mutants and

auxotrophic revertants
Explain the difference between genetic selection and a genetic screen
Design a replica plating experiment that can determine bacterial
genotypes
Diagram conjugation from mating through recombination
Design a blender experiment whose results can be used to create a
genetic map
Be able to define and use the following terms: auxotroph, prototroph,
conditional lethal, reversion, selection, minimal media, bacteria,
clone, replica plating, conjugation, transformation, transduction,
streptomycin, F (Fertility) factor, F+, plasmid, F-, Hfr (High
Frequency of recombination), pilus, exconjugant, E. coli

Lecture 9 4/26/16

Beadle and Tatum - ONE GENE-ONE ENZYME

Factors A, B, C, D control the production of a black pigment. The genes
encoding A, B, C and D are independently assorting. The alternate
alleles that give abnormal function of these genes are a, b, c, and d. A
black AA BB CC DD is crossed with a colorless aa bb cc dd to give a
black F1. The F1 is then backcrossed with a colorless aa bb cc dd.
Assume that A, B, C and D act in a pathway as follows:
colorless

colorless

grey

brown

What percentage of the F2 are brown?

A. 1/2
B. 1/4
C. 1/8
D. 1/16
E. None of above

Lecture 9 4/26/16

D
black

Beadle and Tatum - ONE GENE-ONE ENZYME

Factors A, B, C, D control the production of a black pigment. The genes
encoding A, B, C and D are independently assorting. The alternate
alleles that give abnormal function of these genes are a, b, c, and d. A
black AA BB CC DD is crossed with a colorless aa bb cc dd to give a
black F1. The F1 is then backcrossed with a colorless aa bb cc dd.
Assume that A, B, C and D act in a pathway as follows:
colorless

colorless

grey

brown

What percentage of the F2 are colorless?

A. 1/4
B. 3/8
C. 1/2
D. 3/4
E. None of above
Lecture 9 4/26/16

D
black

Beadle and Tatum - ONE GENE-ONE ENZYME

Factors A, B, C, D control the production of a black pigment. The genes
encoding A, B, C and D are independently assorting. The alternate
alleles that give abnormal function of these genes are a, b, c, and d. A
black AA BB CC DD is crossed with a colorless aa bb cc dd to give a
black F1. The F1 is then backcrossed with a colorless aa bb cc dd.
Genes A, B and C independently assort, but genes C and D are
linked with a 20 map unit separation. Assume that A, B, C and D act
in a pathway as follows:
colorless

colorless

grey

brown

What percentage of the F2 are black?

A. 1/4
B. 1/8
C. 1/10
D. 1/20
E. None of above
Lecture 9 4/26/16

D
black

Various strains of bacteria were incubated on plates containing minimal media

plus several amino acids as indicated. From the pattern of growth, predict
genotypes of colonies 1-4.

arg, met
asp, ile

b
d
arg, met
asp
no ile

b
d

arg, met
ile

arg,
asp, ile

met
asp, ile

no asp

no met

no arg

Which colony (a, b, c or d) has the genotype arg-, met+, asp-, ile+?

Lecture 9 4/26/16

The plates shown below contain different sugars as carbon sources

as indicated. Cells were first plated on minimal media with glucose
(master plate) and then replica plated onto plates with lactose,
mannose or arabinose (no glucose).
e

a
c
b

master plate

lactose

mannose

arabinose

Which colony has the genotype lac- man+ ara+?

Lecture 9 4/26/16

The plates shown below contain different sugars as carbon sources

as indicated. Cells were first plated on minimal media with glucose
(master plate) and then replica plated onto plates with lactose,
mannose or arabinose (no glucose).
e

a
c
b

master plate

a
c

a
c

b
lactose

b
mannose

arabinose

What is the genotype of colony c?

a. lac+ man+ ara+
b. lac+ man- arac. lac- man- ara+
d. lac- man+ ara+
e. None of above
Lecture 9 4/26/16

Hfr strain strs trp+ lys+ his+ is crossed with F- strain strr trp- lys- his-.
You mate and plate on minimal media plus streptomycin and
his:dine and get colonies.
Which genes are selected for on the master plate?

A. Strep and his:dine
B. Strep, tryptophan, lysine and his:dine
C. Strep, lysine and his:dine
D. Strep, tryptophan and lysine
E. None of above

Lecture 9 4/26/16

A strain of E. coli is trp- his- lac+. Which medium would this

bacterium grow on?

A) minimal media with lactose, his:dine, tryptophan
B) minimal media with glucose and his:dine
C) minimal media with maltose and tryptophan
D) minimal media with glucose
E) More than one of above

Lecture 9 4/26/16

how was conjugaGon discovered?

Lets cross two bacteria with complementary genotypes...
met- thr+ x met+ thr-
2x108

108 108

2x108

28

25
MM

24
MM

MM

Reversion happens at a rate of 10-6. Therefore, DNA exchange isnt detectable

over the reversion rate.
Lecture 9 4/26/16
10

how was conjugaGon discovered?

What if we used 4 instead of 2 markers?

met-bio-thr+leu+ x met+bio+thr-leu-
2x108

108 108

2x108

20

0
MM

0
MM

MM

Conclusion When bacteria are mixed, they can exchange genes such that
recombinants can grow on MM.
Lecture 9 4/26/16

11

HfrH strSthr+aziRtonRlac+gal+ x F- strRthr-aziStonSlac-gal-

Bacteria were mixed, at different times they were blended and plated on minimal
plates with strep. This directly selects for Thr+ and Strr colonies. The % of the
unselected donor markers (Azi, Ton, Gal and Lac) were then examined.
Methods and Results

Interpretation
Hfr
9m

10m

16m

25m

3
3
3
3
3
3
3
3

F-

3
3
3
3
3
3
3
3

Plated on MM + streptomycin
Lecture 9 4/26/16

12

HfrH strSthr+aziRtonRlac+gal+ x F- strRthr-aziStonSlac-gal-

interrupted maGng

HfrH x FMix, incubate 15m,
then blend

strR
thr+
azi?
ton?
lac?
gal?

Replica plate

MM + galactose
as only sugar

MM + azide

MM + lactose as
MM + T1 phage
only sugar

Lecture 9 4/26/16

13

HfrH strS thr+ aziR tonR lac+ gal+ x F- strR thr- aziS tonS lac- gal-
What is the genotype of the colony the
red arrow is poin:ng to?

Mix, incubate 15
Then blend.

HfrH x

F-

Minimal Media
+ azide

Replica plate

Minimal Media +
strep + glucose

Minimal Media
+ T1 phage

Strr
Leu+
Thr+
Azi?
Ton?
Lac?
Gal?

Minimal Media
+ lactose as only sugar
source
Lecture 9 4/26/16

A. Azis Tons Lac+ GalB. Azir Tons Lac- GalC. Azis Tonr Lac+ GalD. Azir Tonr Lac- GalE. None of above

Minimal Media
+ galactose as only sugar
source
14

HfrH strS thr+ aziR tonR lac+ gal+ x F- strR thr- aziS tonS lac- gal-
What is the genotype of the colony the
red arrow is poin:ng to?

Mix, incubate 15
Then blend.

HfrH x

F-

Minimal Media
+ azide

Replica plate

Minimal Media +
strep + glucose

Minimal Media
+ T1 phage

Strr
Leu+
Thr+
Azi?
Ton?
Lac?
Gal?

Minimal Media
+ lactose as only sugar
source
Lecture 9 4/26/16

A. Azis Tons Lac+ GalB. Azir Tons Lac- GalC. Azis Tonr Lac+ GalD. Azir Tonr Lac- GalE. None of above

Minimal Media
+ galactose as only sugar
source
15

You have isolated 100 auxotrophic strains in E. coli. None of these 100 strains
can grow on minimal media, but all 100 can grow on minimal media plus
methionine and arginine. Further analysis shows that 30 can grow on minimal
media plus arginine and 20 can grow on minimal media plus methionine. Fill in
the number of strains that have the following phenotype.
met- arg-



met+ arg-



met- arg+



met+ arg+



total

100


How many have a met- arg- genotype?

A. 10
B. 20
C. 30
D. 50
E. Cannot determine, need more informa:on
Lecture 9 4/26/16

16

You have three strains of E. coli, X, Y, and Z.

Mixing X and Z gives no exchange of geneGc markers.
X and Y exchange geneGc markers with a 1 in 104 frequency.
Y and Z exchange geneGc markers at a frequency of 1 in 107.
Label the three strains as F-, F+ or Hfr.

A.

B.

C.

D.

E.

X is:
Y is:
Z is:

Hfr
F+
F-

X is:
Y is:
Z is:

F+
Hfr
F-

X is:
Y is:
Z is:

Hfr
F-
F+

X is:
Y is:
Z is:

F-
Hfr
F+

X is:
Y is:
Z is:

F+
F-
Hfr

Lecture 9 4/26/16

17