Hi everyone,
I'm expressing a protein (more precisely a single-domain antibody) in BL21 (DE3) cells in LB and I should induce with IPTG when the OD600 was between 0.6 - 0.7. But when I checked the OD600 (after 3hrs of incubation at 37ºC, 200rpm) it was 0.9!
What are the implications of that?
PS: For a 500mL culture, I normally add 300uL of IPTG (1M) and then do a O/N over-expression at 16ºC, 200rpm.
Thanks in advance!
Best,
Margarida
I usually induce at OD600= 0.9. However, I have seen some papers say they go until OD600=2!!
I have accidentally let the cells grow to 1.0 and 1.2, and I still got plenty of protein.
I reckon that it strongly depends on the gene product you want to express how important the cell density is - we look at quite a bit of new proteins and usually using OD 0.5-0.6 gives most likely soluble protein expression, if we go over this we may get more protein but not always soluble. In case you get low titers, I recommend to stick to the OD in the protocol and not add more unknowns to the mix.
How much OD you get depends mainly on the oxygenation level. That's given by your RPM, culture volume, flask shape, fill height to volume ratio... In a highly aerated system like a SparkleCell bioreactor, you can easily get OD600 = 15.0 with LB medium and much more with higher nutrient media.
Some of the flask-geometry related details are not usually published, even though they can have rather large effects. Therefore your ODs aren't necessarily comparable with the literature. It can be helpful to express your OD as % of max OD seen in an uninduced control. That way at least your in-house results will become more comparable.
But ultimately there's very little you can do to cope with the lack of published details. Do your own optimizations.
I agree with everyone else here. It usually depends on the product you're trying to get. I'd suggest you try empirically and induce your culture at different stages, say OD 0.6, 0.9, 1.2 and 1.5 and see how it goes. It won't take you long to analyse such result and will save you a lot of time pinpointing the correct answer, where in reality usually empirical personal evidence works best =)
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