Hello In order to know *exactly* what the reason for poor transformation outcome was one has to do all sorts of experiments. This is not likely to be your goal, right? Leaky expression, overload of DNA, somehow compromised cells, or even plain old user error - and numerous other reasons can be proposed (including some esoteric ones like incompatibility of your gene product with a specific strain, for reasons going beyond protocol errors and simple considerations).
This can take a couple of years to sort out. If you're talking about using M15[pREP4] cells then the reason why you failed earlier is likely to be toxicity - pREP4 plasmid supplies lacI in-trans, which represses 'stray' transcription reasonably well. The cells that you used before and did not succeed with must have had low intrinsic levels of lacI and thus permitted leakage. To test this theory you could streak a couple of colonies of the M15[pREP4] cells on a plate that has a small amount of IPTG in the agar (say 0.1 mM) - if the colonies don't grow at all, or appear to be very tiny then you know it's your protein toxicity that's the issue here. Keep in mind that bacteria are extremely survival-oriented and therefore you will eventually generate 'safe' mutants and recombinants that will be able to grow even on IPTG. Likely those mutants will be useless to you from practical standpoint. Once you induce the M15[pREP4] cells - the toxicity will manifest itself again. You will likely observe total cessation of growth (for a long period of time) and possibly even lysis of the culture. Therefore you may have to adjust your fermentation protocol to take this into account. One of the common adjustments is deliberate induction at higher OD, another is to include 0.8% glucose in the growth medium; there are other options available to you. Hopefully once you induce transcription, the gene products do not shut down protein synthesis - which would be a disaster since it would likely shut down its own synthesis as well. Since I don't know what you're growing, this is just one of the possibilities :) Artem > > now i have transformed pqe 30 clone into the m15 host successfully, can > someone let me know exactly what was reason behind the problem into > transformation??is it leaky expression into the other host that is toxic > for the cells,if it so then will i would be able to get good expression > into this host??? > > atul > -----Original Message----- > From: CCP4 bulletin board on behalf of ar...@xtals.org > Sent: Tue 5/5/2009 6:24 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone > > To clarify: I am not implying that I've worked with many proteins that > express better in XL1-blue cells than they would express in BL21(DE3) or > such if cloned into a pET vector or similar. In fact I can probably recall > only one or two that expressed *better* in XL1 cells. However in the good > old days pQE series of vectors was quite commonly used and I had things in > that were inherited from others - these 'things' were fairly simple to > express in XL1-blue whereas they gave me loads of trouble in other strains > and I was too busy/lazy to re-clone them. > > Artem > >> Hello Artem, >> >> We express almost all our proteins in BL21 derivatives. It sounds >> like you've worked with many proteins that express/behave better in >> XL1-Blue? >> >> >> ho >> UC Berkeley >> >> ------------------------------------------------------------------------------------------------- >> XL1-Blue is a strain of E. coli. Whether it is or isn't an expression >> host >> depends on the definition, and I am not going to argue semantics. >> >> The T5 promoter works with regular garden variety RNApol of E. coli. >> Therefore ANY E. coli strain is an 'expression host' for vectors that >> contain this promoter. >> >> I've expressed many proteins in XL1-Blue and I see no reason why you >> can't >> express yours, either. >> >> Artem >> > >
