BIO4320 Mol. Biol. & Genet. Eng. Mid-Term Examination Oct.

29, 2002
Name: Student ID Number
Question 1 (15 marks)

Important note: read all information given to you before answering this question!

Background information:
In the presence of the Cre recombinase, two plasmids carrying the loxP sequence will
undergo site specific recombination to form a co-integrative plasmid (see diagram below).

loxP

C B
A B
loxP Cre recombinase
Co-integrative
plasmid
loxP
C D

D A

loxP

The loxP element is a short (34 bp) DNA sequence that is recognized by the Cre recombinase.
Insertion of such sequnce between a promoter and its target gene usually will not affect gene
expression.

The problem:
You were given Plasmid I (see next page) that contains your target cDNA. You need to test
the function of this cDNA in yeast. However, there is no suitable cloning site for you to
either add a promoter in front of the target cDNA or cut out the target cDNA. You were also
given Plasmid II and Plasmid III that contains a loxP element and a galactose-inducible yeast
promoter, respectivley (see next page).

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 BIO4320 Mol. Biol. & Genet. Eng. Mid-Term Examination Oct. 29, 2002
Name: Student ID Number

KmR

URA3

Plasmid I
Yeast
ori
XhoI-BamHI- EcoRI-HindIII EcoRV
loxP

Target
cDNA
loxP

Plasmid II
BamHI E. coli
ori

ApR

HindIII-SmaI-SalI EcoRI-BamHI-SmaI

ApR: ampillicin resistance gene

CmR: chloramphenicol resistance gene
Galactose-inducible
KmR: kanamycin resistance gene
yeast promoter
URA3: a gene for synthesizing uracil in yeast

Plasmid III
EcoRI E. coli
ori

CmR

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BIO4320 Mol. Biol. & Genet. Eng. Mid-Term Examination Oct. 29, 2002
Name: Student ID Number
The cutting sites of the restriction enzymes are as follows:

BamHI 5’G^GATCC3’
EcoRI 5’G^AATTC3’
EcoRV 5’GAT^ATC3’
HindIII 5’A^AGCTT3’
SalI 5’G^TCGAC3’
SmaI 5’CCC^GGG3’
XhoI 5’C^TCGAG3’

You are also provide with two bacterial strains: (i) TOP10 that carries a gene encoding the
Cre recombinase, and (ii) XL1-Blue that is Cre-free.

Your task is to transfer the galactose inducible yeast promoter from Plasmid III into Plasmid I
so that the promoter is located 5’ to the target cDNA and placed in sense orientation. To do
so, you need to make use of Plasmid II and the Cre-loxP site special recombination system.

Explain your strategy and describe all critical steps (including enzyme cutting, ligation,
choice of bacteria host, antibiotic selection, etc.) with appropriate diagrams.

Your answer should include:

(i) How to cut out the yeast promoter from Plasmid III and subclone it into Plasmid II.
What enzyme(s) to use? What is the appropriate orientation of cloning? Which
bacteria host(s) should be used and how to select successful transformants.
(ii) After subcloned into Plasmid II, how to make use of the Cre-loxP system to place the
yeast promoter to a position so that it can drive the expression of the target cDNA?
Which bacteria host(s) should be used in the process and how to select successful
clones.
(iii) What is the final structure of the co-integrative plasmid? You can use this to check
if you have made any mistakes in the intermediate steps.

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BIO4320 Mol. Biol. & Genet. Eng. Mid-Term Examination Oct. 29, 2002
Name: Student ID Number
Subcloning the yeast promoter into Plasmid II

(i) Digest Plasmid III with SalI (1) and EcoRI (1) to release the galactose-inducible
yeast promoter fragment.
(ii) Digest Plasmid II with XhoI (1) and EcoRI (1).
(iii) Insert SalI-EcoRI promoter fragment into the XhoI and EcoRI cutting sites of
Plasmid II, respectively (1).
(iv) Transform the modified Plasmid II into XL1-Blue (0.5); select the plasmid by
ampillicin (0.5).

Moving the yeast promoter from Plasmid II into Plasmid I

(i) Co-transform (1) Plasmid I and the modified Plamsid II into TOP10 (1).
(ii) Grow in medium containing ampillicin and kanamycin (1) and prepare plasmids
from cells.
(iii) Transformed the plasmid mixture into XL1-Blue (1) to prevent recombination at
loxP sites on the co-integrative plasmid (1). Select by ampillicinand kanamycin
(1).

Final structure of the co-integrative plasmid (5)

Deduct 0.5 mark for each missing piece. Deduct 1 mark for each mis-arrangement.

Target
loxP
Galactose-inducible cDNA
yeast promoter

E. coli KmR

URA3

ApR
Yeast ori

loxP

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BIO4320 Mol. Biol. & Genet. Eng. Mid-Term Examination Oct. 29, 2002
Name: Student ID Number
Question 2 (10 marks)

Assume that you have successfully solved the problem in Question 1. The next step is to
test the function of the target cDNA. DNA sequence analysis shows that the target cDNA
may encode a polypeptide which exhibits high homology to some salt tolerance proteins.
How can you test this hypothesis using a yeast system and the co-integrative plasmid you
constructed in Question 1.

Hint: you need to refer to the features on the DNA constructs (see diagrams in Question 1) to
answer this question.

(i) The co-integrative plasmid resulted has a yeast replication origin (1), a URA3
selectable marker (1), and the expression of the target gene is controlled by a
galactose-inducible yeast promoter (1).
(ii) To begin, first check the lethal dose of salt that will inhibit yeast growth (1).
(iii) Transform the co-integrative plasmid into yeast (1) by methods such as
electrophoration (0.5).
(iv) Use a Ura3 defective yeast mutant as host (1) so that the plasmid can be
selected with the URA3 selection marker (1), by growing the transformed cells
on uracil-free medium (1).
(v) Plasmid-containing cells will be grown on medium containing a lethal dose of
salt (1), in the presence or absent of galactose (1). Galactose will induce the
expression of our target gene (1).
(vi) If the plasmid-containing cells can survive on high salt conditions only in the
presence of galactose, the hypothesis is confirmed (1).

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BIO4320 Mol. Biol. & Genet. Eng. Mid-Term Examination Oct. 29, 2002
Name: Student ID Number
(i)
Question 3 (5 marks)

To clone the promoter of the gene corresponding to the target cDNA in Question 1 and
Question 2, a genomic library was constructed (i.e. a random collection of genomic DNA
fragments were cloned into the multiple cloning sites a vector). The complete sequence of
this vector was known. The DNA sequence of the target cDNA was also determined.
How can you clone its promoter directly by PCR. Describe clearly what primers will be
used and state the approximate locations and appropriate orientations of these primers.

Note: It is not a 5’ RACE experiment, don’t mix up.

One primer site will be chosen based on the cDNA sequence (1). This site will be closed to
the 5’ end (1) of the coding strand (0.5). The primer sequence will be complementary (1)
and runs antiparellelly to the coding strand (1).

Another primer site will be located on the vector (1) closed to the cloning site for the DNA
insert (1). The primer sequence will be pointing to the cloning site (1).

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