I recently got significant functional enzyme produced from a culture of BL21 C41 pLysS cells containing my gene of interest in a pDEST14 vector (the gene codes for a reductase). Functional activity was determined by seeing significant visible degradation of a substrate that I added to the culture upon induction after four days of fermentation at 30C. However, I got similar functional activity in the uninduced control.
Wanting to minimize this leaky basal expression, I repeated the experiment but this time added chloramphenicol (to 34mg/mL to maintain pLysS plasmid) and glucose (to .2% w/v as recommended by the supplier to repress basal expression prior to induction) to the starter culture. Starter was grown overnight and then used at 1:100 the following morning to inoculate two identical flasks, one for the induced culture and the other an uninduced control. No glucose was added to this scale up, but chloramphenicol was added to 34mg/mL to maintain the pLysS plasmid, in addition to the AMP that is used for selection. I induced with IPTG (1mM final at an OD of .8 as recommended by the supplier).
However this time expression became negligible, as seen from minimal/no degradation of the substrate in the culture after four days.
Everything else seems normal; cultures grew at the same rate, were induced at same OD, have remained cloudy, aeration is the same, and all other conditions are the same as the previous experiment.
I expected the expression to be unaffected in the induced culture, since IPTG is added at an appropriate OD and it seems like the glucose would be used up/diluted this point significantly anyway. Can the addition of such a small amount of glucose and chloramphenicol to the starter culture really affect expression this much? Can it delay the expression? Should I have spun down/washed the cells prior to scale up to remove any remaining glucose? Does anyone have any other suggestions for what may be going wrong? Thank you!
Yes I have seen that before. It's not normal. It seems to affect only a few of my constructs. When they see only a small amount of glucose in the beginning, some constructs are extremely unforgiving, and refuse to express even long after glucose is no longer detectable in the medium (you can check easily with a diabetes monitoring kit). So I doubt that washing them would help, but you can try. I don't see any clear pattern as to what might be causing this strange behavior.
As long as you get an acceptable amount of protein out at the other end, I guess don't worry about suppression. With pLysS in there, you should have less of a need for it than you would otherwise.
Madeleine Keefe : In theory it should not matter. Glucose is used for repression because it is a PTS sugar, and so in the presence of glucose the lactose transporter gets phosphorylated and becomes non-functional. That way, unintended induction (due to, say, traces of lactose in tryptone) should be avoided, but induction with IPTG should still work (at 1 mM IPTG enters the cell even in the absence of the lactose transporter). So, at least in BL21(DE3), where T7 RNA pol is under control of the glucose-insensitive lacUV5 promoter, the presence of glucose should not matter. C41(DE3) might be a different kettle of fish, because -if memory serves well- the lac promoter controlling T7 RNA pol there is sensitive to catabolite repression, but I agree with you and John Schloendorn , glucose should be long gone by the time you induce anyway.
But theory is only theory. Can you repeat the experiment, but setting up a parallel control with a starter culture minus glucose? Also, if you do set up that control, can you include two expression cultures inoculated with it, one with and another without chloramphenicol?
Also, from where are you inoculating the starter cultures? From a streaked plate or from aliquots of a cell bank (i.e. glycerol stocks)?
Hope it helps!
Alejandro Martin Thanks for your reply. I did repeat minus the glucose and was still getting not-so-great and pretty inconsistent expression, so I think something else may be wrong.
I have been reading from many sources now that I should not be inoculating from a glycerol stock, as BL21 are apparently notorious for losing/mutating the expression plasmid during storage. I didn't think that this would be too much of an issue since the stocks are brand new (
Madeleine Keefe ,
Loss of expression with time when working with glycerol stocks does happen with BL21(DE3) but in my experience a) it is construct-specific, that is, you get it consistently with some expression constructs but not with others, b) occurs way less frequently than most people claim, and c) is often blamed when the actual cause is that the glycerol stocks were not properly prepared/used.
If your plasmid falls into case a) then using fresh transformation plates does work, but do not fall into the trap of storing the plates in the fridge. If you need to repeat the experiment a week later it's best to repeat the transformation. BL21(DE3) strains transformed with T7 expression plasmids do lose expression when stored at 4*C, see e.g. PMID 15638935.
Also, how are you preparing the glycrol stocks? Just spinning an ON culture and resuspending it into 20-40% glycerol does not cut it and often results exactly in the kind of symptoms you are describing. In my experience it's best to use exponential stage cultures grown in the presence of glucose, and to split the stock into small single-use aliquots stored at -70*C. I have not found flash-freezing into liquid nitrogen to be that important.
Hope it helps!
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