Lecture 1 Exam Questions
Reviewed?
Finished?
Date @September 30, 2025
1. Name two important differences between a bacterial chromosome
and a bacterial plasmid.
Two important differences between a bacterial chromosome and a bacterial plasmid are:
1. Essentiality: A bacterial chromosome contains essential genes. A bacterial plasmid,
however, is a DNA molecule that is not essential for survival under rich defined growth
conditions.
2. Size: A bacterial chromosome is a DNA molecule that is typically larger than plasmids. A
bacterial plasmid is a DNA molecule that is smaller than a chromosome.
2. Give the definition of a bacterial chromosome and a bacterial
plasmid.
Bacterial Chromosome Definition: A bacterial chromosome is a circular DNA molecule
(larger than plasmids) that contains essential genes.
Bacterial Plasmid Definition: A bacterial plasmid is a circular DNA molecule (smaller
than a chromosome) that is not essential for survival under rich defined growth
conditions.
→ Plasmids display huge diversity in terms of sizes, sequences, origin of replication
(incompatibility), selection/counter-selection markers, and GC content.
3. Draw the general organization of a plasmid and name the different
components.
Lecture 1 Exam Questions 1
, A plasmid acts as a cloning vector. While a literal drawing cannot be generated here, the
organization of a general plasmid (like the example pBR322) includes several key
components necessary for its use and expression:
Origin of replication.
Antibiotic resistance genes (e.g., Ampicillin, Tetracyclin).
Restriction enzymes/sites.
Multiple Cloning Site (MCS).
The essential elements needed for the translation and transcription of a gene within the
plasmid (the transcription unit and translation unit) include:
Promoter: Contains sequences like the GACA box (–35 box) and TATA box (–10 box). It
initiates the transcription unit.
Transcription Start Site (+1).
RBS (Ribosome-Binding Site): Also known as the Shine-Delgarno sequence. This
conserved AG-rich sequence is located a few nucleotides upstream of the translation start
site (TSS).
5’ UTR region.
START codon (ATG).
STOP codon (TAA).
3’ UTR region.
Terminator.
Lecture 1 Exam Questions 2
, Omitting even one of these sequences (Promoter, START, STOP, Terminator, and RBS) will
lead to an inability to generate functional transcription.
4. What are restriction enzymes and what is their interest in Gene
Technology?
Restriction Enzymes (Endonucleases):
Restriction enzymes are endonucleases that cut the DNA and generate 3'-OH and 5’-
phosphate ends. They typically recognize sites of 4, 5, 6, or 8 base pairs, which are often
palindromic (due to homo-dimerization). Examples of restriction enzymes include EcoRI,
HindIII, BamHI, SalI, and PstI.
Interest in Gene Technology:
Restriction enzymes are central to the Restriction-ligation cloning method. Their primary
interest is to enable the cloning of a specific DNA fragment into a plasmid. They are used in
the first step of this process to excise the compatible fragment and prepare the insert and
vector for subsequent ligation, forming a recombinant vector.
5. Explain how, using restriction enzymes, we can insert a gene of
interest in the correct orientation into a plasmid.
To insert a gene of interest (GOI) in the correct orientation into a plasmid, it is necessary to
use two different restriction enzymes.
1. These two different enzymes cut the DNA, generating different protruding/sticky ends at
each end of the insert.
2. The vector must also be cut by the same two enzymes, resulting in compatible but
distinct sticky ends for ligation.
3. Since the two ends are distinct, the insert can only ligate into the vector in one specific
way, allowing the specific insertion with the correct orientation.
4. The correct orientation is crucial, especially when the final plasmid includes promoters
and/or tags.
6. What are the advantages and disadvantages of restriction enzymes
that generate blunt or protruding/sticky ends?
Lecture 1 Exam Questions 3
, Feature Protruding/Sticky Ends Blunt Ends
They allow the specific insertion All blunt ends are compatible
Advantage with the correct orientation if with each other (5’-P and 3’-
two different enzymes are used. OH).
No preferential orientation
Ligation requires basic possible (two possibilities for
Disadvantage complementarity between the insertion). Restriction-ligation
protruding ends. using blunt ends can be
inefficient for large inserts.
Restriction enzymes that recognize different sites can sometimes generate compatible
protruding ends for ligation. For example, BamHI (5'-G|GATCC-3') and BglII (5'-A|GATCT-
3') generate compatible extremities.
7. Explain the cloning technique using the Gateway method. Draw.
The Gateway-system is based on the lambda ( λ ) phage recombination system.
Principle and Mechanism:
1. The phage λ infects Escherichia coli (E. coli).
2. Site-specific recombination occurs between the phage attachment site (attP) and the
bacterial chromosomal attachment site (attB). This recombination is catalyzed by Int
(integrase) and IHF (induced host factor).
3. This insertion process (Integration) generates attL and attR sites.
4. The Gateway system uses this mechanism in two main reactions to clone a sequence of
interest:
BP Reaction (Cloning): The gene of interest (flanked by attB sites) is combined
with a donor vector (e.g., pDONR221, which contains attP sites) to create an Entry
vector (which contains attL sites).
LR Reaction (Expression): The Entry vector (with attL sites) is combined with a
Destination plasmid (e.g., pDEST14, which contains attR sites) to generate the final
expression clone.
Key Components/Drawing Description:
The system utilizes attB, attP, attL, and attR sites for specificity.
Lecture 1 Exam Questions 4
Reviewed?
Finished?
Date @September 30, 2025
1. Name two important differences between a bacterial chromosome
and a bacterial plasmid.
Two important differences between a bacterial chromosome and a bacterial plasmid are:
1. Essentiality: A bacterial chromosome contains essential genes. A bacterial plasmid,
however, is a DNA molecule that is not essential for survival under rich defined growth
conditions.
2. Size: A bacterial chromosome is a DNA molecule that is typically larger than plasmids. A
bacterial plasmid is a DNA molecule that is smaller than a chromosome.
2. Give the definition of a bacterial chromosome and a bacterial
plasmid.
Bacterial Chromosome Definition: A bacterial chromosome is a circular DNA molecule
(larger than plasmids) that contains essential genes.
Bacterial Plasmid Definition: A bacterial plasmid is a circular DNA molecule (smaller
than a chromosome) that is not essential for survival under rich defined growth
conditions.
→ Plasmids display huge diversity in terms of sizes, sequences, origin of replication
(incompatibility), selection/counter-selection markers, and GC content.
3. Draw the general organization of a plasmid and name the different
components.
Lecture 1 Exam Questions 1
, A plasmid acts as a cloning vector. While a literal drawing cannot be generated here, the
organization of a general plasmid (like the example pBR322) includes several key
components necessary for its use and expression:
Origin of replication.
Antibiotic resistance genes (e.g., Ampicillin, Tetracyclin).
Restriction enzymes/sites.
Multiple Cloning Site (MCS).
The essential elements needed for the translation and transcription of a gene within the
plasmid (the transcription unit and translation unit) include:
Promoter: Contains sequences like the GACA box (–35 box) and TATA box (–10 box). It
initiates the transcription unit.
Transcription Start Site (+1).
RBS (Ribosome-Binding Site): Also known as the Shine-Delgarno sequence. This
conserved AG-rich sequence is located a few nucleotides upstream of the translation start
site (TSS).
5’ UTR region.
START codon (ATG).
STOP codon (TAA).
3’ UTR region.
Terminator.
Lecture 1 Exam Questions 2
, Omitting even one of these sequences (Promoter, START, STOP, Terminator, and RBS) will
lead to an inability to generate functional transcription.
4. What are restriction enzymes and what is their interest in Gene
Technology?
Restriction Enzymes (Endonucleases):
Restriction enzymes are endonucleases that cut the DNA and generate 3'-OH and 5’-
phosphate ends. They typically recognize sites of 4, 5, 6, or 8 base pairs, which are often
palindromic (due to homo-dimerization). Examples of restriction enzymes include EcoRI,
HindIII, BamHI, SalI, and PstI.
Interest in Gene Technology:
Restriction enzymes are central to the Restriction-ligation cloning method. Their primary
interest is to enable the cloning of a specific DNA fragment into a plasmid. They are used in
the first step of this process to excise the compatible fragment and prepare the insert and
vector for subsequent ligation, forming a recombinant vector.
5. Explain how, using restriction enzymes, we can insert a gene of
interest in the correct orientation into a plasmid.
To insert a gene of interest (GOI) in the correct orientation into a plasmid, it is necessary to
use two different restriction enzymes.
1. These two different enzymes cut the DNA, generating different protruding/sticky ends at
each end of the insert.
2. The vector must also be cut by the same two enzymes, resulting in compatible but
distinct sticky ends for ligation.
3. Since the two ends are distinct, the insert can only ligate into the vector in one specific
way, allowing the specific insertion with the correct orientation.
4. The correct orientation is crucial, especially when the final plasmid includes promoters
and/or tags.
6. What are the advantages and disadvantages of restriction enzymes
that generate blunt or protruding/sticky ends?
Lecture 1 Exam Questions 3
, Feature Protruding/Sticky Ends Blunt Ends
They allow the specific insertion All blunt ends are compatible
Advantage with the correct orientation if with each other (5’-P and 3’-
two different enzymes are used. OH).
No preferential orientation
Ligation requires basic possible (two possibilities for
Disadvantage complementarity between the insertion). Restriction-ligation
protruding ends. using blunt ends can be
inefficient for large inserts.
Restriction enzymes that recognize different sites can sometimes generate compatible
protruding ends for ligation. For example, BamHI (5'-G|GATCC-3') and BglII (5'-A|GATCT-
3') generate compatible extremities.
7. Explain the cloning technique using the Gateway method. Draw.
The Gateway-system is based on the lambda ( λ ) phage recombination system.
Principle and Mechanism:
1. The phage λ infects Escherichia coli (E. coli).
2. Site-specific recombination occurs between the phage attachment site (attP) and the
bacterial chromosomal attachment site (attB). This recombination is catalyzed by Int
(integrase) and IHF (induced host factor).
3. This insertion process (Integration) generates attL and attR sites.
4. The Gateway system uses this mechanism in two main reactions to clone a sequence of
interest:
BP Reaction (Cloning): The gene of interest (flanked by attB sites) is combined
with a donor vector (e.g., pDONR221, which contains attP sites) to create an Entry
vector (which contains attL sites).
LR Reaction (Expression): The Entry vector (with attL sites) is combined with a
Destination plasmid (e.g., pDEST14, which contains attR sites) to generate the final
expression clone.
Key Components/Drawing Description:
The system utilizes attB, attP, attL, and attR sites for specificity.
Lecture 1 Exam Questions 4
