Advanced Centre for Treatment, Research and Education in Cancer
Question
Asked 12 November 2014
What are the ideal conditions for inducing pBAD promoter?
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?
Most recent answer
Hi, Jie Jack Lu
Bacterial cells like E. coli have transcription and translation occurring simultaneously at fast rates. Thus, when a protein is expressed in E. coli, it does not have enough time to fold properly thus causing the formation of aggregates of misfolded proteins. If the same protein were expressed in eukaryotic cells, slower rates of transcription and translation and chaperones would allow proper folding. Thus, to reduce protein misfolding, cells are grown at low temperature (like 18C) to slow metabolic processes including gene expression and protein production. Moreover, at lower temperatures, proteases in E. coli are less active. Overall, low temperature increases success! Jie Jack Lu
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Popular answers (1)
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.
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All Answers (10)
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.
6 Recommendations
University of Paris-Sud
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...
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Catalent Pharma Solutions
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.
University of Paris-Sud
@Jie Jack Lu:
It's to slow down the rate of protein synthesis during induction in order to limit protein misfolding/aggregation.
2 Recommendations
Complutense University of Madrid
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
1 Recommendation
Complutense University of Madrid
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
Tokyo University of Agriculture and Technology
pBAD is recommended for use in ara promoter deficient E. coli like TOP10. Does it still work in BL21?
Advanced Centre for Treatment, Research and Education in Cancer
Hi, Jie Jack Lu
Bacterial cells like E. coli have transcription and translation occurring simultaneously at fast rates. Thus, when a protein is expressed in E. coli, it does not have enough time to fold properly thus causing the formation of aggregates of misfolded proteins. If the same protein were expressed in eukaryotic cells, slower rates of transcription and translation and chaperones would allow proper folding. Thus, to reduce protein misfolding, cells are grown at low temperature (like 18C) to slow metabolic processes including gene expression and protein production. Moreover, at lower temperatures, proteases in E. coli are less active. Overall, low temperature increases success! Jie Jack Lu
1 Recommendation
