Bacterial Transformation Flashcards
(27 cards)
What is the main purpose of the multiple cloning site (MCS) in a plasmid vector?
A) To increase plasmid copy number
B) To enable blue-white screening
C) To allow insertion of foreign DNA at specific sites
D) To confer antibiotic resistance
C) To allow insertion of foreign DNA at specific sites
Q6 (1 mark MCQ)
Why is the pGEM-T Easy vector suitable for T-A cloning?
A) It has blunt ends
B) It contains an antibiotic resistance gene
C) It has 3′ T-overhangs that pair with A-overhangs of PCR products
D) It has a high copy number origin of replication
C) It has 3′ T-overhangs that pair with A-overhangs of PCR products
What is the purpose of colony PCR in a transformation experiment?
To rapidly check if bacterial colonies contain the correct inserted gene without growing them in liquid culture.
Which of the following would indicate successful transformation on an ampicillin plate?
A) A clear zone of inhibition
B) Colonies growing on the plate
C) Blue colonies only
D) No colonies at all
B) Colonies growing on the plate
What are the key features of a plasmid vector used for cloning?
Antibiotic resistance gene: for selection (e.g., AmpR).
Multiple Cloning Site (MCS): contains several restriction sites for inserting DNA.
lacZ gene + promoter: used in blue-white screening.
purpose of blue-white screening?
Distinguishes between recombinant (inserted DNA) and non-recombinant (empty vector) colonies.
White colonies = lacZ disrupted by insert → no β-gal → no blue.
Blue colonies = intact lacZ → β-gal cleaves X-gal → blue.
T-A cloning: why is Taq polymerase used?
adds A overhangs at 3’ ends of PCR product → compatible with T overhangs of vector.
What components are needed in a T-A ligation reaction?
PCR product (with A overhangs)
plasmid vector (with T overhangs)
T4 DNA Ligase
Ligation buffer (contains ATP)
Why is heat shock used in bacterial transformation?
temporarily opens cell membrane via thermal imbalance → allows plasmid entry.
what is the role of IPTG and X-gal in selection plates?
IPTG: induces lac operon → transcription of lacZ if intact.
X-gal: substrate cleaved by β-gal → forms blue pigment.
What are positive and background controls in cloning?
Positive control: uses a known-working insert → confirms transformation setup.
Background control (vector only): detects self-ligation → should only yield blue colonies.
difference between transformation and transfection?
Transformation = introduce plasmid into bacteria (E. coli).
Transfection = introduce plasmid into mammalian cells for gene expression.
After a T-A cloning experiment using pGEM-T and Taq-amplified product, all colonies appear blue. Which is the most likely reason?
A. The ligation failed
B. The competent cells were not viable
C. The insert lacked A overhangs
D. The vector did not contain a lacZ gene
A. The ligation failed
- Blue = intact lacZ → no insert = ligation failed. The insert may have been fine, but without ligation, it never entered the vector.
Which step directly allows you to distinguish between recombinant and non-recombinant colonies on an agar plate?
A. Heat shock
B. IPTG addition
C. Antibiotic selection
D. X-gal conversion
D. X-gal conversion
- X-gal conversion by β-gal leads to blue colour. If insert is present, lacZ disrupted → no β-gal → white.
Why is Taq polymerase specifically compatible with T-A cloning?
A. It has proofreading ability
B. It adds blunt ends
C. It adds A overhangs to PCR products
D. It cuts T overhangs from the vector
C. It adds A overhangs to PCR products
Which of the following would not be expected if you plated only vector without insert on IPTG/X-gal plates?
A. White colonies
B. Blue colonies
C. Self-ligated vector
D. Intact lacZ gene
A. White colonies
you want to express a gene in mammalian cells for 24 hours. What technique do you use?
A. Bacterial transformation
B. Stable transfection
C. Transient transfection
D. T-A cloning
C. Transient transfection
- allows short-term gene expression in eukaryotic cells
What colour colonies indicate successful insertion of a gene in blue-white screening?
white colonies
What component on a plasmid allows antibiotic selection of transformed bacteria?
Antibiotic resistance gene (e.g. AmpR)
Why are IPTG and X-gal included on selection plates in blue-white screening?
IPTG induces expression of the lacZ gene by activating the lac promoter.
X-gal is cleaved by β-galactosidase (lacZ product), forming a blue compound if lacZ is intact.
Define “background control” in bacterial transformation and explain its purpose.
Background control = transformation with vector only (no insert).
Checks for self-ligation events that would produce false positives (blue colonies without insert).
What is the role of the lacZ gene in plasmid vectors?
encodes β-galactosidase, which cleaves X-gal to form a blue product.
disruption by an insert leads to white colonies in blue-white screening.
Describe the steps involved in transforming competent E. coli cells with a recombinant plasmid.
Thaw competent cells on ice to preserve membrane integrity.
Add recombinant plasmid DNA to the cells.
Heat shock at 42°C to create a temporary pore in the membrane → DNA enters.
Recover in SOC media → allow expression of the antibiotic resistance gene.
Plate on antibiotic agar (antibiotic equivalent to resistance gene) → only transformed cells survive and grow.
List and explain four key features of a plasmid vector used in T-A cloning.
Antibiotic resistance gene – allows selection of transformed bacteria.
lacZ gene – used in blue-white screening to detect inserts.
T overhangs – enable ligation with PCR products that have A overhangs.
Origin of replication (ori) – ensures plasmid replication in host cells.
