Download Solutions to 7.014 Problem Set 3

Solutions to 7.014 Problem Set 3
Question 1
You are studying the bacterium Escherichia coli (E.coli). You have two different strains of E.coli.
Strain A can grow on minimal media and is sensitive to the antibiotic, ampicillin. Strain B
requires supplemented media but has the ability to grow on plates containing ampicillin.
Strain A is:
Arg+ Ura+ Leu+ His+ Gly+ AmpS
Strain B is:
Arg- Ura- Leu- His- Gly- AmpR
a) One afternoon someone accidentally mixes your two strains together.
i) How could you purify Strain A away from Strain B?
If you grow the mixture on plates that contain minimal media, only the Strain A cells will grow.
ii) How could you purify Strain B away from Strain A?
If you grow the mixture on plates that contain fully supplemented media that contains ampicillin
only the Strain B cells will grow.
You want to map the order of the genes on the E.Coli chromosome. You perform timed mating
experiments in which you disrupt mating at different time points. You test the resulting
bacteria on plates lacking one of the following: Arg, Ura, Leu, His, or Gly.
b) Which strain would you use as the donor? Which strain would you use as the recipient?
Why?
You would use Strain B as the recipient and Strain A as the donor. Strain A has wild-type, functioning
copies of the genes required to make Arg, Ura, Leu, His, or Gly. As these genes are passed from Strain
A to Strain B, Strain B will sequentially acquire the ability to make these compounds. You can see this
by the recipient strain's ability to grow in media missing one of these supplements.
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Question 1, continued
You do the experiment and obtain the following data. (+) indicates the resulting bacteria
grow, (-) indicates the resulting bacteria fail to grow.
Time (Minutes)
10
20
30
40
50
- ARG
+
+
+
+
Complete media, fully supplemented except….
-URA
-LEU
-HIS
-GLY
+
+
+
+
+
+
+
+
+
+
+
c) What is the order of markers on the bacterial chromosome?
Question 2
You have decided to study the biosynthesis of tryptophan in Saccharomyces cerevisiae (yeast) for
your senior project. You want to create tryptophan auxotrophs by mutagenesis of a wild-type
strain of yeast.
a) Would you begin with a haploid strain or a diploid strain? Why?
You would begin with a haploid strain. A haploid strain carries a single copy of each gene. When you
mutagenize haploid cells, a single mutation can result in a phenotype, even when the phenotype is
recessive. In a diploid, you would need to mutate both copies of a gene to pick up a recessive phenotype.
b) You mutate the wild-type strain such that each cell has only a single mutation. How would
you identify the tryptophan auxotrophs among your mutant strains?
You would grow your mutant cells on fully supplemented media. You would then replica plate those
mutants to media that lacked only trytophan. Cells that grow on fully supplemented media but not on
media that lack trytophan are likely your tryptophan auxotrophs.
c) After identifying a set of mutants (trp 1-7), you perform a complementation test . How is a
complementation test performed?
A complementation test is performed by making diploid cells using pairwise combinations of
the haploid mutant. The resulting diploid
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Question 2, continued
Data from the complementation test shows:
trp 1
trp 2
trp 3
trp 4
Wt
+
+
+
+
trp 1
+
+
trp 2
+
+
+
trp 3
+
+
trp 4
+
+
+
trp 5
+
+
+
trp 6
+
+
+
trp 7
-
trp5
+
+
+
+
+
-
trp 6
+
+
+
+
+
-
trp 7
-
+ indicates growth and - indicates no growth.
d) From the data above, list the complementation groups that are represented. How many
genes have you identified in your screen?
e) The trp 7 mutant has what kind of phenotype? Can you assign trp 7 to a complementation
group?
You repeat this mutagenesis and screen many times and characterize the resulting mutants.
You discover that this biosynthetic pathway involves intermediates T, U, V, W, and X and the
genes Trp a - Trp e. You make a master plate of mutants Trp a - Trp e. Using the replica
plating technique you screen these mutants on different plates containing an intermediate or
tryptophan. The following is a representation of your data.
Master Plate
minimal medium + trp
b
a
c
d
e
b
d
d
minimal
+T
c
minimal
+U
d
c
minimal
+V
b
c
d
b
a
c
d
e
e
minimal
+W
minimal
+X
f) Indicate the order of intermediates and enzymes for the tryptophan biosynthetic pathway.
______
à
______
à
______
à
______
à
______
à
tryptophan
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Question 3
A mutant hunt is performed in yeast to elucidate the biosynthetic pathway of adenine
production. Adenine production is involved in yeast cell color. If there is a mutation that
disrupts progression of the pathway the yeast cell has a color other than the usual white color.
The mutants from the screen represent the five enzymes that are involved in the biosynthesis
of adenine. The phenotype of each mutant is listed below.
Mutant
Mut A
Mut B
Mut C
Mut D
Mut E
Phenotype
yellow
pink
red
orange
green
a) You decide to perform an epistasis test.
i) How is an epistasis test done?
ii) What information do you get from such a test?
The following are the results from the double mutant experiments.
Wild type
Mut A
Mut B
Mut C
Mut D
Mut E
Wild type
White
yellow
pink
red
orange
green
Mut A
yellow
yellow
pink
yellow
yellow
yellow
Mut B
pink
pink
pink
pink
pink
pink
Mut C
red
yellow
pink
red
orange
green
Mut D
orange
yellow
pink
orange
orange
orange
Mut E
green
yellow
pink
green
orange
green
b) Assuming that there is one linear biosynthetic pathway involved in adenine production,
what is the order of this pathway?
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Question 4
As extra credit for 7.014 you want to reproduce the Griffith experiment. Instead of
Streptococcus pneumoniae you use the agent that causes mad cow disease. You isolate two forms
of the agent, one form is indeed disease causing, the other is harmless.
1) You take a sample of disease causing agent, inject it into a mouse, and the mouse dies.
2) You take a sample of the harmless agent, inject it into a mouse, and the mouse lives.
3) You take a sample of disease causing agent, heat kill this agent, inject it into a mouse, and
the mouse lives.
a) If your experiment is to duplicate the Griffith experiments,
i) What would be your next experiment (experiment 4)?
ii) If the agent causing Mad Cow disease behaves like Streptococcus pneumoniae, What do
you predict happens in experiment 4?
ii) What conclusions were drawn from Griffith's experiments?
b) To further study the agent that causes Mad Cow disease, you treat the disease-causing agent
in different ways and then assay the ability of the agent to kill injected mice.
Treatment
Heat kill sample
Add DNAse
Add protease
Fate of mouse injected
with treated sample
lives
dies
lives
i) What do you think the causative agent of Mad Cow disease is? Why?
ii) Would this disease be inherited?
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