I am studying in vivo expression using pBAD18 Cm vector in BL21 cell strain. I am confused about the induction procedure. I am using 0.2% arabinose to induce pBAD promoter at 0.2 OD growth.
Can I induce the cells at lower OD value? Will there be any toxic impact on the cells if I chose to induce at lower OD?
Hi Gaurav
While using pBAD promoter for protein purification, we use 0.1% arabinose for induction. You can induce at lower O.D. as well, we induce at O.D. of 0.1, and this works for everyone in our lab. Also, there's no toxicity whatsoever.
So if you're setting up 1 liter culture for protein purification, add 0.1% arabinose after 1 hour of inoculation.
Best regards with your experiments.
Hi Gaurav
While using pBAD promoter for protein purification, we use 0.1% arabinose for induction. You can induce at lower O.D. as well, we induce at O.D. of 0.1, and this works for everyone in our lab. Also, there's no toxicity whatsoever.
So if you're setting up 1 liter culture for protein purification, add 0.1% arabinose after 1 hour of inoculation.
Best regards with your experiments.
Hi there,
There is no ideal condition for pBAD induction! Depending on what you want and what you actually get, you can adjust/play with OD of induction, concentration of arabinose, duration of induction and temperature of induction...
It is said that induction should be carried out at higher OD if the protein is toxic to the cell. Lower OD induction will make the cells vulnerable to toxicity.
@Jie Jack Lu:
It's to slow down the rate of protein synthesis during induction in order to limit protein misfolding/aggregation.
It depends what you want kind of gene you want to induce. If it's for purification of the protein or just to see the effect of the gene. I usually use pBAD to induce potential resistance gene. To do so, I use MH-broth supplemented with 1% of L-arabinose. I usually induce for at least one hour and a half after reaching the exponential phase
It depends what you want kind of gene you want to induce. If it's for purification of the protein or just to see the effect of the gene. I usually use pBAD to induce potential resistance gene. To do so, I use MH-broth supplemented with 1% of L-arabinose. I usually induce for at least one hour and a half after reaching the exponential phase
pBAD is recommended for use in ara promoter deficient E. coli like TOP10. Does it still work in BL21?
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