lab quiz 1 Flashcards
purpose of this lab
study the function of a gene and elucidate the biochemical pathways in which it operates
the protein encoded by our gene can be produced in…
E. coli
expression steps
- transform E. coli
- grow to mid-log phase
- induce with IPTG
- continue growth, then harvest cells
- SDS PAGE analysis
the E. coli strain used depends n
plasmid and expression considerations
Bl21
E. coli strain used for protein expression
Bl21 (DE3)
E. coli strain used for protein expression
BL21-CodonPlus(DE3)-RIL strain
E. coli strain used for protein expression
may be used to express genes that have rare codons
expression of genes in E. coli that use “rare” codons may result in…
low levels of expression
transform E. coli and grow to mid log phase:
steps in the curve
lag phase, exponential (log) phase, stationary phase, death phase
optical density at ___ may be used instead of __________.
A600
cell number
optimization parameters for protein expression
- OD (optical density) at induction
- IPTG amount added
- induction time
- incubation temp for induction
- host strain used
lyse cells and purify protein by affinity chromatography
- Lyse harvested cells
- purify expressed proteins by affinity chromatography
- analyze by SDS page
resin and buffer used for affinity chromatography depend on…
the protein tag in the recombinant protein
ArcticExpress strains express
recombinant proteins
How do we use IPTG as an inducer?
removes a repressor from the lac operon to induce gene expression
