Solution Key - 7.013 Exam 2 (4/3/13) - With Answers Page 5
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7Question 4 continued
c) You then plan to amplify the recombinant plasmid in E. coli bacterial cells. You transform the E. coli
with the ligation mix and plate them on a master plate (growth medium with no antibiotics). You then
replica- plate the colonies on plate 1 (growth medium + ampicillin) and plate 2 (growth medium containing
both ampicillin and kanamycin). You obtain the following colonies.
i.
Circle the plate (1 or 2) in the schematic
that contains bacterial colonies that have
the recombinant plasmid.
ii.
In the plate that you circled, fill in/ color
the colonies that contain the recombinant
plasmid. Explain why you selected these
Plate 2
Plate 1
colonies.
R
R
The recombinant plasmid will have an intact and functional Amp
gene but a disrupted Kan
gene where Gene A
R
S
is inserted. So any bacterial cell that will receive the recombinant plasmid will be Amp
Kan
and will therefore
grow on plate 1 but not on plate 2.
d) Another group is competing with you. Although they have the bacterial clone that contains the
recombinant plasmid with Gene A in correct orientation they cannot express Gene A in bacteria. To
understand what the issue is they decide to PCR amplify and sequence Gene A isolated from their
bacterial clone (mutant) and compare it with the sequence of Gene A that you published (wild- type).
Shown below is the sequence flanking the mutant allele of Gene A.
To p strand
5’GAAATC
GGACTT3’
G ene A
Gene A
Bo ttom strand
3’CTTTAG
CCTGAA5’
They have the following primers for amplifying Gene A by PCR reaction. Circle the primers that they
would use to PCR amplify Gene A.
#1:
5’GAAATC3’
#2: 5’TTCAGG3’
#3: 5’CTTTAG3’
#4:
5’AAGTCC3’
e) Sequencing results show that the mutant version has one point mutation compared to the wild- type
version of Gene A. Shown below is a portion of the fluorescence dideoxy- sequencing gel that gives the
sequence of the mRNA like strand/ non-template strand of the DNA that corresponds to amino acids
5-7.
Fluorescent dideoxy- C
3’
i.
Write the sequence of the mRNA
Fluorescent dideoxy- G
that corresponds to amino acids 5-7
Fluorescent dideoxy- T
of the mutant allele of Gene A.
Fluorescent dideoxy- A
5’AUGUAGAUG3’
5’
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ii.
If the wild –type allele has the amino acids N- met
-trp
-met
-C, circle the base in the sequence
that you gave in part (i) that has undergone point mutation in the mutant allele. Note: Codon
chart is provided on the last page. You can detach the last page.
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5’UGG3’ (coding for trp
) is changed to 5’UAG3’ which is a stop codon
iii.
Circle the type of mutation that you see in the mutant allele from the choices below (choose fro
m
silent, missense,
nonsense
or frameshift).
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