Question about protein expression induction - (Jan/21/2006 )
Hi, I have a question about proteni expression.
When expressing recombinant proteins in E.coli, most protocols suggest to grow the E. coli culture to about 0.7-0.8 then induce with IPTG. Why 0.7??? Can I grow the culture to 1.5-1.6 then induce?? Will it matter? What's the theory behind this?
And also, how would you determine the time for induction? Is two hour induction is appropriate for most of E. coli cultures?
I would appreciate it if someone can help me out with this.
I've been doing this sort of thing with IPTG for years now... too many years.
You want to induce at a range of 0.4-0.7 OD 600 because that is when the bacteria are in log growth. They are at their healthiest and most viable stage of their lives... ideal for protein expression.
If you wait until somthing like 1.5 for an OD, then they are past log growth.. many are dying and thus unable to express your protein of interest.
The way to go about this is as such: Assume you want a 1l flask of bacteria to produce your protein of interest. The night before, pick a single colony from a streak to isolation of bacteria and culture in 25 ml of media. The next day, subculture 20ml of the bugs into the 1l flask. Moniter growth by taking a 1ml sample every 30 min and reading the OD. When the OD is at 0.5 A 600 then add the IPTG and allow it to express for 2-6 hrs. Then harvest the bacteria.
The above is just a general idea of what needs to be done. You may need to tweak time and OD or volume of bacteria to get the optimal protein expression.
i know its too late to say that but
i was looking for an answer like that.