Download 1 - UCSF Biochemistry

Bacterial Problem Set
Problem 1:
λ lysogens are immune to superinfection by λ, but not with other lambdoid phages
such as 434. λimm434 (a lambdoid hybrid phage that has mostly λ genes except
for cI and the surrounding “immunity” region of 434 phage) also can grow on E.
coli(λ).
a. If you take a 1ml culture of E. coli(λ) (this is wild-type E. coli) and superinfect
with λimm434 at an MOI of 1, the tube will clear after about an hour. Describe
how you would determine whether the released phages from this infection are
λimm434 or λ (induced from the prophage). Do you expect there will be more
λimm434, more λ, or equal numbers of both phages in the lysate?
b. You perform infection experiments using wild-type E. coli and E. coli(λ),
infecting them at an MOI = 1 with either λ, 434, λimm434, or λimm434 Ram (an
amber mutant in the R gene) and get the following result:
λ
E. coli
+
E. coli(λ)
+ = lysis, - = no lysis
Infecting Phage
434
λimm434
+
+
+
+
λimm434 Ram
+
Explain, in detail, why λimm434 Ram is able to lyse the λ lysogen but not wild-type
E. coli. (about 4-6 sentences)
c. Do you expect there are viable phages in the lysate from the λimm434 Ram
infection of E. coli(λ) and, if so, what phage genotype will be present?
Bacterial Problem Set
Problem 2:
As mentioned in class, a number of interesting alleles of the genes in the SOS pathway in E. coli
have been identified. Describe how you would screen/select for:
a. alleles of recA that are normal at 32C but activated at 42C (recAtif, tif = temperatureinduced filamentation)
b. alleles of lexA that are uninducible (lexAind) by UV
c. lexA alleles that are defective (lexAdef – lexA is an essential gene). You can assume any
molecular genetic tool is available (except access to the recA and lexA alleles in question,
of course).
For each, describe a secondary test you would perform that would increase your confidence that
you have the desired allele. Indicate whether your approach is a screen or selection, and whether
you think each resulting allele is dominant or recessive.
Bacterial Problem Set
Problem 3:
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
13
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
2
3
4
WT
+
+
+
+
cI+
NA
+
-
cII+
+
NA
+
cIIINA
+
+
+
Bacterial Problem Set
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.
Bacterial Problem Set
Problem 4:
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
three strains and get the following results:

phage stocks
(host strain)


(K-12)
Skyler
Jesse
(Marie)
(Hank)
K-12
+
+
+
Skyler
+
+
+
+
+
(+ = huge plaque, - = none or few single plaques)
Test Strain
Jesse
+
-
Marie
+
+
+
Hank
-
Bacterial Problem Set
a. 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?).
b. 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’
cys
62’
61’
recA
45’
his
trp
Bacterial Problem Set
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:
media:
His- MetHis- LeuHis- Lac+
His- Gal+
His- Trpmating
time
(min)
0
1
2
3
4
+
5
+
10
+
15
+
+
20
+
+
25
+
+
+
30
+
+
+
+
35
+
+
+
+
40
45
50
55
60
i. Which strain is the donor strain and which is the recipient?
ii.
From these results, can you determine if the donor molecule is an F’ plasmid or an Hfr
chromosome?
iii.
Assuming the donor strain is Hfr, draw a map showing the position of the integrated
plasmid and indicate the direction of transfer.
iv.
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.
v.
Design a scheme that would allow you to create the his+ trp+ strain.