Expression of a possible toxic gene?
Posted 30 November 2009 - 07:38 AM
The nature of the protein is slightly dubious, all I can say is its fairly closely related to phospholipase A2 in snake venom and therefore I suspect it may be toxic to E.coli, I monitored the growth curves of the induced E.coli culture and found that post induction (at around 0.6 O.D) the culture stagnated reaching only 0.7 compared to a non-induced protein growth curve which reached 1.4 in the same time frame.
However my supervisor has told me that all induced cultures stop growing post induction (little change at O.D 600), I guess normally its something I would never have bothered to check but i'm quite supprised that this occurs. Any ideas?
Any advice would be much appreciated, cheers!
Posted 30 November 2009 - 08:58 AM
the only thing I can suggest is to incubate at a much lower temp (if you haven't already) down to RT.
also, if it's being produced at all, you'll see it before the culture dies. have you tried a quick-and-dirty prep with SDS page of culture fractions? this will tell you if it's produced at all. I would try many timepoints, including a pre-induction sample for comparison. even if you don't see a band, if it's toxic in small amounts you may need to do a Western or something on your extracts.
PCR and RD notwithstanding, I'd take a few days and get it sequenced. you could have a point mutation or a frameshift or something that's hindering transcription
Posted 30 November 2009 - 09:00 AM
Posted 30 November 2009 - 09:12 AM
aimikins, good idea with the quick dirty preps, I check my samples on SDS PAGE after about 3 hours induction using a cracking buffer. But i will have a check after half a hr next time at a much earlier time-frame. Sequencing is not a option saddly because of the cost but I did manage to 'borrow' some high performance, proof-reading polymerase so I think its probably unlikely to be a frameshift or point mutation present.
I his-taged it to aid in purification but also so I could do a western to check it out (i have the antibody) but our development fluid has run out so I'll have to wait a while till the order forms go through to check that out. I've tried expressing at temperatures as low as 20 degrees aswell.
The biggest worry I have is that the growth stagnates immediatly after induction but my supervisor is convinced that this isnt a issue at all so maybe i'm just being stupid
Posted 30 November 2009 - 09:52 AM
Just my .02.
Posted 30 November 2009 - 02:14 PM