Flashcards in Making an expression construct Deck (8)
What are some advantages to using E. coli?
Easy and quick to grow to high cell densities
Easy to clone
Highly developed plasmid vectors
Well understood genetics
What are some disadvantages to using E. coli?
Requires a complex media
Poor protein secretion
Not used industrially
Lacks some auxiliary biochemical pathways
Not good at protein folding
Describe transcriptional fusion
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
Describe translational fusion
The join between the promoter and inserted DNA lies after the translation start
High protein production
Allows N-terminus modification
Construct has to be arranged to place inserted sequence in frame with the start codon
What are inducible promoters?
A promoter that is 'off' until a sufficient cell density has been reached and then expression is induced
Describe IPTG induction
Lacz is present on the E. coli chromosome and produces mRNA encoding β-galactosidase
Expression is induced by inducers of the lac operon (IPTG)
What are fusion tags?
Regions of coding sequences supplied by the expression vector which are expressed in frame with the desired polypeptide