Examenvragen 2026 Gene Technology Fie Thijs
Examenvragen Gene technology
CHAPTER 1
Noem 2 belangrijke verschillen tussen een bacterieel chromosoom en een bacterieel plasmide.
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.
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.
Geef de definitie van een bacterieel chromosoom en plasmide.
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.
Teken de algemene organisatie van een plasmide en noem de verschillende componenten.
A plasmid acts as a cloning vector. 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 GACA (–35) and TATA (–10). It initiates the transcription unit.
- Transcription Start Site (+1).
- RBS (Shine-Delgarno seq.) = conserved AG-rich sequence located few nucleotides upstream of TSS
- 5’ UTR region.
- START codon (ATG).
- STOP codon (TAA).
- 3’ UTR region.
- Terminator.
1
,Examenvragen 2026 Gene Technology Fie Thijs
Wat zijn restrictie-enzymen en wat is het nut ervan in Gentechnologie ?
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.
Leg uit hoe we met de hulp van restrictie-enzymen een gen in de juiste oriëntatie kunnen klonen
➔ Necessary to use two different restriction enzymes.
1. These 2 different enzymes cut DNA, generating different protruding/sticky ends at each end of insert.
2. Vector must also be cut by the same 2 enzymes = compatible but distinct sticky ends for ligation.
3. The two ends are distinct = insert can only ligate into vector in 1 specific way
-> specific insertion with the correct orientation.
Wat is het voor- en nadeel van restrictie-enzymen die stompe of uitstekende uiteinden genereren?
Feature Protruding/ sticky ends Blunt ends
+ They allow the specific insertion All blunt ends are compatible with each other
with the correct orientation if 2 (5’-P and 3’-OH).
different enzymes are used.
- Ligation requires basic No preferential orientation possible (2 possibilities for
complementarity between the insertion). Restriction-ligation using blunt ends can be
protruding ends. inefficient for large inserts.
Leg de kloneringstechnieken uit met de Gateway-methode. Teken.
The Gateway-system is based on the lambda ( λ ) phage recombination system.
1. The phage λ infects Escherichia coli (E. coli).
2. Site-specific recomb. occurs between phage attachment site (attP) and bacterial chromosomal
attachment site (attB). 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:
o BP Reaction (Cloning): GOI (flanked by attB sites) is combined with a donor vector (which contains
attP sites) = create Entry vector (contains attL sites).
o LR Reaction (Expression): Entry vector (with attL sites) is combined with Destination plasmid (which
contains attR sites) = generate final expression clone.
2
,Examenvragen 2026 Gene Technology Fie Thijs
Leg de kloneringstechnieken uit met de Infusion/Gibson-methode. Teken.
The Gibson Assembly method is based on the In-Fusion principle. It is highly efficient, easy, and allows for
one-step targeted cloning of any gene of interest into any vector at any locus. It is seamless (leaves no
disruptive "scar" sequence), ensuring translational reading frame continuity.
In-Fusion Cloning:
-15–20 bp overlap at ends of cloning inserts and linearized cloning vector
(or adjacent inserts if multiple fragments are joined).
- In-Fusion enzyme mix generates single-stranded 5' overhangs at the
terminus of vector and insert. These overhangs then anneal at the sites of
complementarity.
Gibson Cloning Mechanism
1. Exonuclease Activity: 5'-3' exonucleases create single-stranded 3' overhangs.
2. Annealing: Complementary sequences (the 15–20 bp overlaps) then merge.
3. Extension and Sealing: DNA polymerase extends the 3' ends, and DNA ligase seals remaining nicks.
4. Result: The result is a completely sealed double-stranded DNA (dsDNA) molecule.
3
, Examenvragen 2026 Gene Technology Fie Thijs
Leg de kloneringstechnieken uit met de Golden Gate-methode. Teken.
1. Enzyme Type: Golden Gate uses Type II restriction
enzymes
2. Enzymes cut DNA and produce 4 bp sticky ends next
to binding sites, regardless of adjacent nucleotide
sequence. The binding sites of Type IIs restriction
enzymes are notably not palindromic.
3. Assembly Preparation (Drawing Description):
-> Vector contains two BsaI recognition sites in
opposite directions.
-> Restriction enzyme recognition sites incorporated
into insert DNA fragments using PCR, ensuring the
correct orientation.
4. One-Tube Assembly: The PCR fragments and the vector are mixed in a one-tube reaction containing a
type IIs enzyme + T4 DNA ligase.
5. Assembly: Cutting with BsaI exposes complementary sequences for fragment assembly. DNA ligase
seals the nicks. Because the restriction site itself is cleaved and removed, the assembled final product
does not contain the original restriction sites.
Leg de kloneringstechnieken uit met de Slice-methode. Teken.
SlICE = Seamless Ligation Cloning Extract. It is a cheaper alternative to commercially available DNA
assembly kits, such as Gibson Assembly, which typically have a high cost per reaction.
1. Extract Basis: The technique is based on bacterial extracts only.
2. Recombination System: It uses a single in vitro recombination
reaction powered by the lambda ( λ ) phage recombinase. Specifically, it
utilizes the optimized λ prophage Red recombination system, PPY.
3. Components: Cell extracts from E. coli (e.g., the PPY strain carrying
the lambda recombinase) are used.
4. Assembly: DNA fragments in vitro with short end overlaps. This
involves an exonuclease for the double-stranded DNA ends, and a
single-strand annealing protein (Beta) which binds the resulting 3’
overhang and anneals it to a complementary strand.
4
Examenvragen Gene technology
CHAPTER 1
Noem 2 belangrijke verschillen tussen een bacterieel chromosoom en een bacterieel plasmide.
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.
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.
Geef de definitie van een bacterieel chromosoom en plasmide.
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.
Teken de algemene organisatie van een plasmide en noem de verschillende componenten.
A plasmid acts as a cloning vector. 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 GACA (–35) and TATA (–10). It initiates the transcription unit.
- Transcription Start Site (+1).
- RBS (Shine-Delgarno seq.) = conserved AG-rich sequence located few nucleotides upstream of TSS
- 5’ UTR region.
- START codon (ATG).
- STOP codon (TAA).
- 3’ UTR region.
- Terminator.
1
,Examenvragen 2026 Gene Technology Fie Thijs
Wat zijn restrictie-enzymen en wat is het nut ervan in Gentechnologie ?
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.
Leg uit hoe we met de hulp van restrictie-enzymen een gen in de juiste oriëntatie kunnen klonen
➔ Necessary to use two different restriction enzymes.
1. These 2 different enzymes cut DNA, generating different protruding/sticky ends at each end of insert.
2. Vector must also be cut by the same 2 enzymes = compatible but distinct sticky ends for ligation.
3. The two ends are distinct = insert can only ligate into vector in 1 specific way
-> specific insertion with the correct orientation.
Wat is het voor- en nadeel van restrictie-enzymen die stompe of uitstekende uiteinden genereren?
Feature Protruding/ sticky ends Blunt ends
+ They allow the specific insertion All blunt ends are compatible with each other
with the correct orientation if 2 (5’-P and 3’-OH).
different enzymes are used.
- Ligation requires basic No preferential orientation possible (2 possibilities for
complementarity between the insertion). Restriction-ligation using blunt ends can be
protruding ends. inefficient for large inserts.
Leg de kloneringstechnieken uit met de Gateway-methode. Teken.
The Gateway-system is based on the lambda ( λ ) phage recombination system.
1. The phage λ infects Escherichia coli (E. coli).
2. Site-specific recomb. occurs between phage attachment site (attP) and bacterial chromosomal
attachment site (attB). 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:
o BP Reaction (Cloning): GOI (flanked by attB sites) is combined with a donor vector (which contains
attP sites) = create Entry vector (contains attL sites).
o LR Reaction (Expression): Entry vector (with attL sites) is combined with Destination plasmid (which
contains attR sites) = generate final expression clone.
2
,Examenvragen 2026 Gene Technology Fie Thijs
Leg de kloneringstechnieken uit met de Infusion/Gibson-methode. Teken.
The Gibson Assembly method is based on the In-Fusion principle. It is highly efficient, easy, and allows for
one-step targeted cloning of any gene of interest into any vector at any locus. It is seamless (leaves no
disruptive "scar" sequence), ensuring translational reading frame continuity.
In-Fusion Cloning:
-15–20 bp overlap at ends of cloning inserts and linearized cloning vector
(or adjacent inserts if multiple fragments are joined).
- In-Fusion enzyme mix generates single-stranded 5' overhangs at the
terminus of vector and insert. These overhangs then anneal at the sites of
complementarity.
Gibson Cloning Mechanism
1. Exonuclease Activity: 5'-3' exonucleases create single-stranded 3' overhangs.
2. Annealing: Complementary sequences (the 15–20 bp overlaps) then merge.
3. Extension and Sealing: DNA polymerase extends the 3' ends, and DNA ligase seals remaining nicks.
4. Result: The result is a completely sealed double-stranded DNA (dsDNA) molecule.
3
, Examenvragen 2026 Gene Technology Fie Thijs
Leg de kloneringstechnieken uit met de Golden Gate-methode. Teken.
1. Enzyme Type: Golden Gate uses Type II restriction
enzymes
2. Enzymes cut DNA and produce 4 bp sticky ends next
to binding sites, regardless of adjacent nucleotide
sequence. The binding sites of Type IIs restriction
enzymes are notably not palindromic.
3. Assembly Preparation (Drawing Description):
-> Vector contains two BsaI recognition sites in
opposite directions.
-> Restriction enzyme recognition sites incorporated
into insert DNA fragments using PCR, ensuring the
correct orientation.
4. One-Tube Assembly: The PCR fragments and the vector are mixed in a one-tube reaction containing a
type IIs enzyme + T4 DNA ligase.
5. Assembly: Cutting with BsaI exposes complementary sequences for fragment assembly. DNA ligase
seals the nicks. Because the restriction site itself is cleaved and removed, the assembled final product
does not contain the original restriction sites.
Leg de kloneringstechnieken uit met de Slice-methode. Teken.
SlICE = Seamless Ligation Cloning Extract. It is a cheaper alternative to commercially available DNA
assembly kits, such as Gibson Assembly, which typically have a high cost per reaction.
1. Extract Basis: The technique is based on bacterial extracts only.
2. Recombination System: It uses a single in vitro recombination
reaction powered by the lambda ( λ ) phage recombinase. Specifically, it
utilizes the optimized λ prophage Red recombination system, PPY.
3. Components: Cell extracts from E. coli (e.g., the PPY strain carrying
the lambda recombinase) are used.
4. Assembly: DNA fragments in vitro with short end overlaps. This
involves an exonuclease for the double-stranded DNA ends, and a
single-strand annealing protein (Beta) which binds the resulting 3’
overhang and anneals it to a complementary strand.
4
