Making an expression construct

Question 1

What are some advantages to using E. coli?

Answer 1

Easy and quick to grow to high cell densities
Easy to clone
Highly developed plasmid vectors
Well understood genetics

Question 2

What are some disadvantages to using E. coli?

Answer 2

Requires a complex media 
Poor protein secretion
Not used industrially 
Lacks some auxiliary biochemical pathways 
Not good at protein folding

Question 3

Describe transcriptional fusion

Answer 3

The join between the promoter and inserted DNA lies between the transcription and translation start
Easy to make construct
The protein sequence is the same as in original source
Low protein production
modification of N-terminal signal sequence and tags is difficult

Question 4

Describe translational fusion

Answer 4

The join between the promoter and inserted DNA lies after the translation start
Favoured
High protein production
Allows N-terminus modification
Construct has to be arranged to place inserted sequence in frame with the start codon

Question 5

What are inducible promoters?

Answer 5

A promoter that is ‘off’ until a sufficient cell density has been reached and then expression is induced

Question 6

Describe IPTG induction

Answer 6

Lacz is present on the E. coli chromosome and produces mRNA encoding β-galactosidase
Expression is induced by inducers of the lac operon (IPTG)

Question 7

What are fusion tags?

Answer 7

Regions of coding sequences supplied by the expression vector which are expressed in frame with the desired polypeptide

Question 8

What do fusion tags do?

Answer 8

The polypeptide has the functional properties of the target protein and tag and the tag is used to detect/purify the expressed protein or direct it to a cellular location
Tags are designed to be removed from a purified protein by proteases