Prokaryote Problem Set
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Please be explicit and detailed in your answers in order to receive full credit.
Please type out answers (you can draw in any diagrams).
Have your name, question #, and page number (example: John Smith, Question
#1, Page 1 of 2) on each page.
Start each question on a new page and staple each question together separately.
1. You perform a screen for new clear plaque mutants in , you mutagenize WT:GFP (GFP is
inserted into a non-essential region of the  genome) with EMS and plate on WT K12 E. coli,
and screen for clear plaques. Out of 10,000 plaques, you find 50 clear mutants. These mutants
fall into four complementation groups:
Comp. Group
1
2
3
4
#mutants
11
15
11
12
Since you know that cI-, cII-, and cIII-mutants form clear plaques, you perform
complementation tests with one mutant from each complementation group with known  mutants
(no GFP), assaying for cloudy plaques. (+ = cloudy plaques, lysogen formation)
1
2
3
4
WT
+
+
+
+
cI+
+
+
cII+
+
+
cIII+
+
+
a. What gene is likely mutated in the mutants of complementation groups 1-3?
b. From this analysis, can you determine if any of the mutations are dominant or recessive?
You isolate multiple lysogens from each of the plaques in the complementation tests, colony
purify, and do two experiments. First, you test for GFP expression and get the following results.
(w = all white colonies, g = all green colonies, g/w = mix of green and white colonies)
WT cIcIIcIII1
2
3
4
g/w
w
g/w
g/w
g
NA
g
g
g/w
w
NA
g/w
NA
w
g/w
g/w
You then treat each lysogen with UV light to test for prophage induction. Here are the results (+
means phages produced):
1
WT
+
cI+
cII+
cIIINA
Prokaryote Problem Set
2
3
4
+
+
+
NA
+
-
+
NA
+
+
+
+
c. Why are all the lysogens produced from co-infections with cI- green?
d. Posit an explanation for why no phages were produced from the lysogens produced from the
co-infections of 4 + cI-? What gene is likely mutated in 4? Describe an experiment to test
this.
2. After getting shut out in your first quarter rotation picks, you end up in the laboratory of a
new UCSF professor, Dr. Walter White. For your rotation project, the cunning but mad PI hands
you four mysterious vials labeled Skyler, Jesse, Marie, and Hank. Dr. White claims each
contains one of four strains of E. coli but none of them are  lysogens. Just to be sure, you
attempt to confirm that no prophages exist by performing the following three experiments: 1.
You treat a culture of each strain with UV light and test for prophage induction, and 2. You
perform PCR on chromosomal DNA from each strain using oligos specific to  genes. 3. You
spot 105 wild-type  particles (from a stock grown on wild-type E. coli K-12) in the middle of a
lawn of each strain to test for immunity. The results are as follows:
Expt. 1 and 2
Strain
Prophage induction
Skyler
Jesse
Marie
Hank
+
PCR product
+
Expt. 3
Strain: Skyler
huge plaque
Jesse
few single plaques
Marie
Hank
huge plaque
no plaque
Finally, you make stocks of phage grown on each strain (for the Hank strain, you use the phage
induced after UV treatment) and test all of your phages for growth on new lawns of K-12 and the
four strains and get the following results:
Prokaryote Problem Set

phage stocks
(host strain)


(K-12)
Skyler
Jesse
(Marie)
(Hank)
K-12
+
+
+
Skyler
+
+
+
+
+
Test Strain
Jesse
+
-
Marie
+
+
+
Hank
-
(+ = huge plaque, - = none or few single plaques)
Did Dr. White lie to you? Give a reasonable explanation for the differences between the five E.
coli strains (i.e. what didn’t Dr. White tell you about the strains?).
3. Suppose Dr. White gives you the following additional information about the genotypes of
Hank and Skyler:
Hank = leu+, lac+, gal+, bio-, trp+, his-, recA-, cys+, met+
Skyler = leu-, lac-, gal-, bio+, trp-, his+, recA+, cys-, met(lac and gal are required for growth on plates containing either lactose (Lac) or galactose (Gal) as
the sole carbon source, respectively, and the others are auxotrophic markers). White says the only
way he will write a positive rotation evaluation is if you are able to do the impossible, make a
his+ trp+ strain. Luckily, you packed your handy E. coli chromosome map with the position of
these markers indicated (the entire chromosome is 100 min.):
leu lac
met
91’
gal
2’ 8’
bio
17’
17.5’
28’
62’
61’
cys
recA
trp
45’
his
After mixing cells of strains Hank and Skyler together, you find met+ his+ cells at high frequency.
You surmise that one of your strains is an Hfr strain and you perform a disrupted mating
experiment and plate the cells on different types of media. You get the following interesting
results, where + indicates the growth of at least one colony on the indicated media:
Prokaryote Problem Set
media:
mating
time
(min)
0
1
2
3
4
5
10
15
20
25
30
35
40
45
50
55
60
His- Met-
His- Leu-
His- Lac+
His- Gal+
His- Trp-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
-
+
+
-
-
a. Which strain is the donor strain and which is the recipient? From these results, can you
determine if the donor molecule is an F’ plasmid or an Hfr chromosome? If not, what
experiment would you do to determine this?
b. Assuming the donor strain is Hfr, draw a map showing the position of the integrated
plasmid and indicate the direction of transfer.
c. Propose a reasonable explanation for why no recombinants were ever found after 35 min
or longer of mating and why no Trp+ His+ recombinants were ever obtained. How would
you test this idea?
d. Design a scheme that would allow you to create the his+ trp+ strain.