Mapua University BE176 Cellular and Molecular Bioengineering

Module 1 Practice Exam

Question 1 Why? In a cDNA library, the cloned DNA represents

only the expressed genes (mRNA) from the source
Put the following steps in the appropriate order for organism. Each mRNA molecule is reverse-
constructing a genomic library: 1. Ligate the vector transcribed into a complementary DNA (cDNA)
and insert DNA together using DNA ligase. 2. Isolate strand corresponding to a single gene. Therefore, each
genomic DNA from organism. 3. Transform a vector in a cDNA library typically carries the cDNA
population of host cells (E. coli). 4. Cut both the of only one gene. In contrast, genomic libraries contain
genomic DNA and the chosen vector with the same fragments of the entire genome, which can include
restriction enzyme(s). 5. Plate the transformed cells multiple genes or non-coding regions within a single
onto selective media. cloned fragment.
A. 1-2-3-4-5 Question 4
B. 2-1-4-3-5
C. 2-4-1-3-5 Unlike a genomic DNA library, particular care must
D. 3-5-1-2-4 be taken to ensure that the cDNA library represents
E. 4-1-3-2-5 every gene in the genome. Why is this the case?

Question 2 A. A cDNA library lacks introns.

B. Host cells are more likely to reject vectors
You plan to create an E. Coli genomic DNA library with cDNA as compared to genomic DNA.
and clone a gene for arginine synthesis (the ARG1 C. It is harder to clone cDNA fragments as
gene) by complementation of a mutant phenotype. compared to genomic fragments into the
Which of the following statements is true? 1. You vectors.
would isolate genomic DNA from a wild type cell. 2. D. Some genes are transcribed only in certain
You would isolate genomic DNA from an ARG1- cell types or at low level
mutant cell. 3. You would transform a population of
wild type host cells. 4. You would transform a Why? In a cDNA library, only the genes that are being
population of ARG1- mutant cells. 5. You would plate actively transcribed into mRNA at the time of
the transformed cells onto media with ampicillin. 6. extraction will be included. This means that certain
You would plate the transformed cells onto media genes might not be represented if they are not being
without arginine. expressed in the specific cell type or under certain
conditions. Additionally, some genes are transcribed at
A. All of these statements are true. very low levels, making it harder to capture their
B. 1,4,5 cDNA.
C. 1,4,6
D. 2,3,5 Question 5
E. 2,3,6
Select the true statement or statements.
Question 3
A. Restriction enzymes break a phosphodiester
A recombinant vector in a yeast genomic library may bond on only one of the two DNA strands.
contain more than one gene. This is not the case with B. Restriction enzymes break a phosphodiester
the recombinant vectors in a yeast cDNA library bond on both of the DNA strands.
because C. All restriction enzymes produce a region of
single-stranded DNA.
A. different restriction enzymes are used. D. Two different linear DNA molecules are cut
B. every cDNA fragment cloned into each with a single restriction enzyme such that
vector carries only one gene. only one end of each molecule is cut and
C. the host cells cannot be transformed with a these ends have regions of single stranded
vector carrying more than one gene. DNA. The two different DNA molecules will
D. the vectors used in making a cDNA library have compatible ends.
are different. E. Two different linear DNA molecules are each
cut with a different restriction enzyme such
Mapua University BE176 Cellular and Molecular Bioengineering

that only one end of each molecule is cut and DNA. The two different DNA molecules will
these ends have regions of single stranded NOT have compatible ends.

Question 6

You have isolated many different yeast auxotrophs. You want to make a yeast genomic library so that you can clone
all of the genes identified by these mutants. You begin with two different yeast strains, strain 1 and strain 2, each of
which fails to grow in the absence of lysine. You want to use your library to clone the gene or genes that can restore
the yeast to lysine prototrophy.

A. When you construct the yeast genomic library in E. coli, what type of yeast would you choose as the donor
for the genomic DNA?
Use a wild-type yeast strain to complement the mutated vectors in order to restore the lysine prototrophy.

Why? By using wild-type yeast genomic DNA, your library will contain the correct, functional versions of
the genes involved in lysine biosynthesis. When you introduce this library into the auxotrophic strains, the
functional lysine biosynthesis gene from the wild-type yeast can restore lysine prototrophy in the mutants.

B. Once the DNA is isolated from the yeast cells, what would be your next step in preparing the DNA for use
in constructing a library?
The next step is restriction digestion, where the vector and DNA are cleaved using the same restriction
enzyme to complement one another.

C. You successfully prepare yeast genomic DNA and need to choose a vector that will allow you to…
• Insert the yeast genomic DNA fragment, transform E. coli with the ligation mix, select for E. coli cells
that have obtained a plasmid, and obtain colonies of these cells.
• Isolate library clones from E. coli and use them to transform yeast.
• Clone by complementation the gene or genes that can restore the yeast to lysine prototrophy.
You need to choose a vector that will allow you to complete this experiment. List the minimum features
that are required in your vector to execute the plan outlined above.
• The vector must not contain a lysine biosynthesis gene since we are aiming to create a functional
complementation procedure.
• The vector should have multiple cloning sites to facilitate the insertion of yeast.
• The vector should have an origin of replication.
• The vector should have a selectable marker that harbors antibiotic resistance.

D. You digest both the yeast genomic DNA and many copies of the vector with the BamH1 restriction
enzyme. You mix the genomic fragments with the cut vectors and add DNA ligase. You then transform E.
coli cells with the ligation mix. What growth medium can you use to distinguish the bacterial colonies that
carry a vector from the ones that do not carry a vector?

The growth medium should be Lys- medium. The growth of colonies would represent the
transformants of the culture since they grew even without the presence of an essential growth factor.
Mapua University BE176 Cellular and Molecular Bioengineering

E. You successfully create a yeast genomic library in E. coli cells. Outline how you would use this library to
clone by complementation the gene that can restore the yeast lysine prototrophy in strain 1.
• Prepare the yeast strain for transformation:
o You need to use yeast strain 1, which is lysine auxotrophic (i.e., cannot grow without lysine).
o Grow the auxotrophic yeast strain in a medium supplemented with lysine to ensure the cells
are healthy before transformation.
• Isolate the yeast genomic library from E. coli:
o Isolate the plasmid DNA from the E. coli cells that carry the yeast genomic library. This will
include the plasmids containing different yeast genomic DNA fragments.
• Transform yeast strain 1 with the plasmid library:
o Use a yeast transformation protocol (such as the lithium acetate method or electroporation) to
introduce the plasmid library (which contains the yeast genomic DNA fragments) into yeast
strain 1.
o Some yeast cells will take up plasmids carrying different fragments of yeast genomic DNA,
potentially including the gene that complements the lysine auxotrophy.
• Select for transformed yeast cells:
o Plate the transformed yeast cells onto selective media lacking lysine (e.g., a synthetic defined
(SD) medium without lysine).
o Only yeast cells that have taken up a plasmid containing a functional gene involved in lysine
biosynthesis will grow on this medium. These cells have been "complemented" by the
plasmid-borne gene, restoring lysine prototrophy.
• Isolate and analyze complementing yeast clones:
o Isolate individual yeast colonies that grow on the lysine-deficient medium.
o These colonies likely contain the plasmid carrying the gene that restored lysine biosynthesis
in strain 1.
• Recover plasmids from the yeast cells:
o Extract plasmid DNA from the yeast cells that grew on the lysine-deficient medium. You can
do this using a yeast plasmid extraction protocol.
• Re-transform E. coli with the recovered plasmid** (optional step to amplify the plasmid):
o Transform E. coli with the recovered plasmids to amplify and maintain the plasmid.
o Plate the E. coli on LB agar containing the appropriate antibiotic to select for colonies that
carry the plasmid.
• Identify the gene:
o Sequence the yeast genomic DNA fragment in the plasmid to determine which gene from the
genomic library complemented the lysine auxotrophy.
o Alternatively, you could perform a restriction digest or PCR analysis if you know the
approximate size or sequence of the candidate genes involved in lysine biosynthesis.

F. Will the clone identified in part (e) also be able to restore the yeast lysine prototrophy in strain 2? Explain.

It depends on whether strain 1 and 2 are affected on the same genes that affected their lysine biosynthesis.
If they have that same mutated gene, the clone in part e would work. However, if they are different, you need to
start from the DNA genomic library again.
Mapua University BE176 Cellular and Molecular Bioengineering

G. Do you expect that the library you created can be used to restore an arginine auxotroph to arginine
prototrophy? Explain.

It may have the potential to restore a yeast auxotroph that does not produce arginine provided that
the genomic library contains the arginine biosynthesis gene.

Question 7

A schematic of the vector p7012 is shown. The restriction enzymes listed cut only where indicated; they do not cut
anywhere else in the vector or insert.

A. A schematic of gene W is below. You want to clone all of gene W DNA into the p7012 vector. Give three
different strategies that you could use to clone gene W into p7012 and obtain colonies that contain a
recombinant plasmid.

• Strategy 1 uses the restriction enzyme(s) Kpn1 to cut the vector and restriction
enzyme(s) Kpn1 to cut Gene W
• Strategy 2 uses the restriction enzyme(s) Sal1, EcoR1 to cut the vector and restriction
enzyme(s) Xho1,EcoR1 to cut Gene W
• Strategy 3 uses the restriction enzyme(s) BamH1 to cut the vector and restriction
enzyme(s) BamH1 to cut Gene W

B. In which of the strategies would Gene W be inserted into the vector in only one direction?

Strategy 2 will restrict Gene W to be inserted into the vector in one direction. This is because we
used different enzymes to cleave both ends of the vector and gene, which would make the gene unidirectional
as it will complement the end that the same restriction enzyme was cut.
Mapua University BE176 Cellular and Molecular Bioengineering

Question 8

You purify a protein from a plant cell that can act as a potential appetite suppressant. Owing to its possible
commercial application you decide to clone the gene, Gene A, that encodes this protein. You isolate this gene from
the plant cell, clone it into a plasmid vector and amplify it in the bacterial cells.

A. List three features that a plasmid must have to allow the cloning and amplification of Gene A in bacterial
cells.
• Origin of replication.
• Selectable biomarker for antibiotic resistance.
• Restriction sites similar to Gene A.

B. You decide to use the following plasmid to clone Gene A. To achieve this you digest both the genomic
DNA and plasmid DNA using a restriction enzyme. You then ligate the Gene A DNA into the cut plasmids.
Finally, you transform the E. coli bacterial cells with the ligation mix (the recombinant plasmids). Note:
The recognition sites for Kpn 1 and Sal1 on plasmid are 1 kb apart.

Which restriction enzyme (Kpn I, Hind III, Sal I or Xho I) did you use to digest Gene A for
insertion in to the plasmid? Kpn1
Which restriction enzyme (Kpn I, Hind III, Sal I or Xho I) did you use to digest the plasmid
before insertion of Gene A? Briefly explain why. Kpn1, since it will promote complementation of the
foreign DNA and vector.

C. You then plate these transformed bacterial cells onto media that will allow you to distinguish between
bacterial cells that obtained the plasmid and those that did not. Onto what type of growth medium will you
plate your transformation mix? Explain your answer.

The growth medium will contain the antibiotic ampicillin. This will select the bacterial cells that
took up the recom. DNA since it has the AmpR gene, harboring ampicillin resistance. Those cells that did
not take up the vector will not grow.

Question 9
Mapua University BE176 Cellular and Molecular Bioengineering

Name one advantage and one disadvantage in using phage vectors in transforming eukaryotic cells.

• Phage vectors are typically used for transforming bacterial cells and have limitations when it comes to
transforming eukaryotic cells. However, some phage vectors are used in certain eukaryotic transformation
systems, such as bacteriophage lambda vectors in yeast.
• Advantage:
- High Efficiency of Insertion: Phage vectors, particularly bacteriophage lambda, can efficiently
package and deliver large DNA fragments. This high efficiency in DNA insertion and delivery can
be advantageous when working with large genes or complex genetic constructs.
• Disadvantage:
- Limited Host Range: Phage vectors are generally adapted for bacterial cells, and their use in
eukaryotic cells is limited. For eukaryotic transformation, the phage vector must be engineered to
be compatible with eukaryotic cellular machinery, which can be complex and challenging. This
limited host range makes phage vectors less versatile for eukaryotic cell applications compared to
vectors specifically designed for eukaryotic cells, such as yeast or mammalian expression vectors.

Question 10

Explain the results of a blue-white screen. How can you utilize the ampicillin resistance gene in this screening
strategy?

In a blue-white screening strategy, E. coli cells transformed with a cloning vector are plated on agar
containing ampicillin and X-gal. The vector includes a lacZα gene fragment, which encodes β-galactosidase that turns
blue when cleaving X-gal. If the vector contains no insert, the lacZα gene is functional, and colonies turn blue. When
the vector has an insert, the lacZα gene is disrupted, preventing β-galactosidase production, resulting in white colonies.
The ampicillin resistance gene in the vector is used to select for cells that have taken up the vector, ensuring only
transformants grow on the ampicillin-containing medium. Thus, the blue-white screen helps identify recombinant
colonies with the desired insert.