Lecture 3

Question 1

What plasmid should be used

Answer 1

Bacterial plasmids (circular) 6-12 kbp

Question 2

What are the typical features of cloning plasmids

Answer 2

• ORI
 ̶  initiation of DNA replication
• Gene for selection
 ̶  antibiotic resistance (e.g. ampR)
• Multiple cloning sites (MCS)
 ̶  region where foreign DNA can be inserted
• Unique RE
 ̶  RE sites for restriction mapping (Expt 4)
• LacZ gene* 
– blue/white selection

Question 3

What is the use of a promoter

Answer 3

Promoter: allows efficient
transcription of the inserted gene
(drives transcription)

Question 4

What is Polylinker/MCS

Answer 4

Site of insertion
for gene of
interest

Question 5

What is the use of transcription termination sequence

Answer 5

Can improve stability of mRNA and protein yield

Question 6

What is the use of selectable genetic marker

Answer 6

Selectable marker allows selection of cells containing the recombinant expression vector

Question 7

What is a operator

Answer 7

permits regulation
through a specific repressor that
will bind to it

Question 8

What is the use of Ribosome binding site

RBS

Answer 8

provides
sequence signals
required for efficient
translation of mRNA
derived from the
inserted gene

Question 9

How does DNA ligase operate

Answer 9

Catalyzes formation of phosphodiester
bond between 3'‐hydroxyl and 5'‐
phosphate of adjacent bases:
• ATP‐dependent
• 3 main steps

Question 10

What are the 3 main step in ligase

Answer 10

1) adenylylation
(addition of AMP)
of lysine in the
active site,
releasing
pyrophosphate

2) Activation of 5’ phosphate in nick
transfer of AMP to 5’
phosphate to form
pyrophosphate bond

3) Displacement of AMP seals nick

Formation of a
phosphodiester bond
(5' phosphate of the
donor and the 3'
hydroxyl of the
acceptor), release of
AMP

Question 11

What is Transfection (or transformation)

Answer 11

“The process of introducing DNA into cells”

Question 12

What bacterium is considered transformed?

Answer 12

A bacterium carrying introduced

plasmid DNA

Question 13

What are the two ways of transformation of plasmid DNA into bacteria

Answer 13

 Heat shock method (also: CaCl2 method)

 Electroporation method

Question 14

What are the steps Heat Shock method

Answer 14

1) Mix ‘competent’ bacterial cells and recombinant plasmid in solution with CaCl2
2) Chill on ice (plasmid will adhere to outside of bacterial cell)
3) Incubate cells briefly in a waterbath at 42°C. Plasmid DNA will enter cells through pores created in the bacterial cell
4) Plate out transformation mixture on LB/Amp plate and incubate at 37°C for 24 hr
5) Only transformed cells will express gene for antibiotic resistance and will grow on LB/Amp plate

Question 15

What are the steps for Transformation via Electroporation method

Answer 15

1) Mix bacterial cells and recombinant plasmid

2) Transfer to electroporation
cuvette

3) Apply brief electrical shock (e.g. 1200 V for 3 ms)
4) Plate out transformation mixture on LB/Amp plate and incubate at 37°C for 24 hr
5) Only transformed cells will express gene for antibiotic resistance and will grow on LB/Amp plate

Question 16

How do you determine the result of the ‘transformed’ cells containing plasmid?

Answer 16

• Untransformed E. coli are ampicillin sensitive  they do not grow

• Transformed E. coli containing plasmids
(white colonies) are ampicillin resistant

Question 17

What should you do when you propagate microorganisms?

Answer 17

Aseptic technique (also sterile technique):
• procedure that is performed under sterile conditions
• must always be used when preparing growth media (liquid media
or agar plates) for bacterial propagation

Question 18

How do you propagate microorganisms?

Answer 18

•Inoculate growth medium (such as Luria broth) containing antibiotic
(e.g. ampicillin) with positive clone (cells containing the recombinant DNA)

• Incubate at 37 C in aerated shaker incubator u
• Nutrients in growth medium
• Antibiotic used in the growth medium

•Only cells containing the recombinant DNA plasmid (antibiotic resistance
gene) will grow

Question 19

CONFIRMATION OF RECOMBINANT PLASMID

Answer 19

DNA SEQUENCING

Sanger sequencing method
‐ Developed by Frederick Sanger in 1977
‐ Uses PCR methodology
‐ Cyclic amplification
‐ Relies on ddNTP chain termination