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expression problems?


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#1 Cindy

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Posted 21 October 2004 - 10:00 AM

I have cloned a gene sequence from a pUSE vector into a pRSETB vector and transformed into BL21 cells. I go to do a small scale protein expression experiment and I run into a few problems.

1. If I innoculate a 25 ml culture with 4 ml of overnight culture and add the selective antibiotics, the OD for the culture acutally decreases over the course of 4 hours of incubation and shaking in a 37 degree incubator. I can never get it above .1
I hesitate to try to induce with IPTG if the OD is so low. If I take a 1 ml sample out of the 25 ml culture and spin it down, the pellet is almost nonexistent.

2. If I don't use antibiotics before I induce, I get a great OD reading after 4 hours of incubation. I induce and take 1 ml samples out every hour to lyse the bacteria and look at protein expression. I run the samples out on a 15% SDS PAGE gel and either Coomassie stain or Sypro Orange stain (and have even western blotted for the 6X His tag in the RSET vector) and there is a bunch of protein expression in the uninduced sample and the protein expression lessens as time of induction goes on.

What in the world could be wrong with all of this? Any suggestions would be greatly appreciated.

#2 Oleksii

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Posted 25 October 2004 - 07:40 AM

Did you see any OD in your overnight culture with antibiotic? If not, I think you protein toxic for E.coli.

Alexei

#3 Cindy

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Posted 25 October 2004 - 08:04 AM

The overnight culture was very cloudy and looked as though it had grown well overnight. I didn't check the OD on it though.

#4 Oleksii

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Posted 28 October 2004 - 05:40 AM

Hello Cindy,

I am sorry, first time I misunderstood you. So, without antibiotics you have strong expression even before induction. It nothing unusual, you promoter leeks. But way you have nothing with antibiotic?.. What I can assume, you protein interfere somehow with selective marker in your vector. What kind of protein you express? You can choose anther vector with different selective marker. But if you protein expressed well without antibiotic and you can isolate it do, you really need to change anything?
My notes probably not helpful :lol: . Sorry


Alexei




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