Why does the addition of IPTG allow induction of protein expression?
Unlike lactose, IPTG is not part of any metabolic pathways and so will not be broken down or used by the cell. This ensures that the concentration of IPTG added remains constant, making it a more useful inducer of the lac operon than lactose itself.
How does IPTG function to induce protein expression?
IPTG or Isopropyl β-D-1-thiogalactopyranoside is a chemical reagent mimicking allolactose, which removes a repressor from the lac operon to induce gene expression. It acts as an inducer to initiate the transcription of genes in the lac operon.
What is induced protein?
In the strict sense induction is defined as an enhanced rate of transcription. In drug metabolism the term is used to describe an increased amount of enzyme protein regardless of the underlying mechanism. An enhanced stability of the protein and/or mRNA are included in this loose definition.
What concentration of IPTG is used for induction?
IPTG is an effective inducer of protein expression in the concentration range of 100 μM to 1.0 mM. Concentration used depends on the strength of induction required, as well as the genotype of cells or plasmid used.
How long is IPTG induction?
*Induction times vary from 2-5hrs. *IPTG can be varied from 0.1-1.0M. *If you boil your sample too long they will become viscous from total release of cellular DNA. You can still use them if you can find an area of low viscosity, however, its usually better just to repeat the experiment.
Why BL21 is used for protein expression?
The BL21(DE3)pLysS competent cells provide tighter control of protein expression for expression of toxic proteins and are resistant to chloramphenicol. When used with the CE6 bacteriophage, the BL21 cells provide the tightest control of protein expression (see BL21(DE3) Strains and Protein Toxicity).
How do you stop IPTG induction?
It is possible to partially (because IPTG is non-metabolizable) cease the protein expression by removing IPTG and resuspending cells in fresh medium but then it can also be done by storing the cells at 4C or below. Perhaps certain protein synthesis inhibitors such as Rifamycin or Tetracycline could also be used.