Lecture 42. Techniques 2: Cloning

Question 1

How can you express a eukaryotic gene in E. coli?

Answer 1

By converting the mRNA of the subject into cDNA (complementary DNA)

Question 2

How can a gene with a known sequence be cloned?

Answer 2

By performing PCR with primers with engineered restriction sites

Question 3

Synthesis of the first strand of cDNA

Answer 3

1. Hybridise a poly(dT) primer to the polyA tail in the mRNA
2. Use an RNA-dependent DNA polymerase called reverse transcriptase to make a single strand DNA copy of the mRNA
3. Remove mRNA strand by alkaline digestion

Question 4

Synthesis of the second strand of cDNA

Answer 4

4. Add oligo(dG) to 3’ end of cDNA using Terminal Transferase
5. Hybridise oligo (dC)
6. Use DNA polymerase to make a second DNA strand

Question 5

What is special about Type II restriction enzymes?

Answer 5

They are used extensively for DNA manipulations

Question 6

How many types of restriction enzymes are there?

Answer 6

4

Question 7

What do Type II restriction enzymes recognise?

Answer 7

A 4, 6 or 8 bp sequence known as a restriction site

Question 8

After recognition of a restriction site, what does the Type II restriction enzyme do?

Answer 8

It hydrolyses a phosphodiester bond in each strand of the DNA

Question 9

What are most Type II restriction sites?

Answer 9

Palindromic

Question 10

What are plasmids used for in coding?

Answer 10

Vectors for inserting our genes

Question 11

What does an origin of replication (ori) do?

Answer 11

This allows the plasmid to be maintained in the host cell
Different vectors have different ‘copy numbers’

Question 12

What does a selectable marker (e.g an antibiotic resistant gene) do?

Answer 12

When plated in the presence of an antibiotic, only those bacteria containing the plasmid will grow

Question 13

What does a disruptable marker (e.g lacZ’) do?

Answer 13

This allows selection of recombinant plasmids with DNA cloned into the disruptable marker

Question 14

What does a no conjugation ability do?

Answer 14

The plasmid vector cannot easily be spread from cell to cell – it can only be transmitted by cell division, forming a clone

Question 15

How does pUC cloning work?

Answer 15

Cut plasmid with enzymes and insert correct plasmid using DNA ligase (recombinant plasmid with disrupted lacZ’)
Rapidly growing E. coli treated with CaCl₂ will take up plasmids

Question 16

How does CaCl₂ treatment and heat shock work?

Answer 16

The surface of E. Coli is negative and outer LPS membrane is phosphorylated.
The divalent cation might shield the negatively charged DNA allowing adhering to the outer membrane.

The heat-pulse may create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall

Question 17

What does the process of electroporation involve and what does it do?

Answer 17

DNA can be pushed into a bacterial cell with an electric pulse (10-20 kV/cm)
This is thought to create holes (pores) in the bacterial membrane, allowing plasmid DNA to enter
The pores are then healed rapidly by membrane-repair mechanisms

Question 18

How does colour selection of recombinants work?

Answer 18

Colonies that still contain lacZ’ will form a blue dimer whilst colonies without lacZ’ will appear white as no complementation could take place.

Question 19

Expressing cDNA in E. coli

Answer 19

Ligate your cDNA into a special plasmid, an expression plasmid. This contains all required elements for transcription and translation. Can make lots of protein easily

Question 20

How can a gene/protein be tagged?

Answer 20

After performing PCR with primers that also encode restriction enzyme sites for cloning, insert a hexa-histidine tag followed by a STOP codon

Question 21

What can a hexa-His tagged protein bind to?

Answer 21

Nickel

Question 22

How can a hexa-His tagged protein be purified?

Answer 22

1. Express hexa-His tagged protein in E. coli
2. Extract proteins
3. Load complex mix of proteins on to nickel-agarose column
4. Wash
5. Elute hexa-His tagged protein with imidazole
6. Dialyse for pure protein

Question 23

What are fusion proteins?

Answer 23

Two proteins fused together that combine the activities of the two parental proteins