Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Animal and Molecular Model Systems > Microbiology Forum
Register Search Today's Posts Mark Forums Read

Microbiology Forum Discuss Microbiology Science and Protocols here. Post questions on the study of viruses, fungi, parasites and bacteria here. Microbiology Forum.


Trying to explain odd effect observed in recombinant E.Coli

Trying to explain odd effect observed in recombinant E.Coli - Microbiology Forum

Trying to explain odd effect observed in recombinant E.Coli - Discuss Microbiology Science and Protocols here. Post questions on the study of viruses, fungi, parasites and bacteria here. Microbiology Forum.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-22-2009, 11:56 AM
Pipette Filler
Points: 872, Level: 16 Points: 872, Level: 16 Points: 872, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Estonia
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Default Trying to explain odd effect observed in recombinant E.Coli



Hello,

I've usually induced my recombinant protein in E.Coli with 0,5 mM IPTG when A600 has reached 0,5-0,6. However last time I accidentally let the culture grow to 1,0 and the results have been confusing. It seems that I have got an almost 200% increase in the amount of my recombinant protein. I can't find any articles explaining this as according to my understanding when A600 has reached 0,9 the cells enter stationary phase of growth and recombinant yields should drop when inducing. Has anybody got an explanation or ideas? I would be very grateful.
Reply With Quote
  #2  
Old 02-22-2009, 06:29 PM
Ph.D Doctorate
Points: 3,253, Level: 37 Points: 3,253, Level: 37 Points: 3,253, Level: 37
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2008
Posts: 154
Thanks: 0
Thanked 17 Times in 16 Posts
Default Re: Trying to explain odd effect observed in recombinant E.Coli

What is the protein and why should its synthesis parallel the growth curve?
Reply With Quote
  #3  
Old 02-23-2009, 12:17 PM
Pipette Filler
Points: 872, Level: 16 Points: 872, Level: 16 Points: 872, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Estonia
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Trying to explain odd effect observed in recombinant E.Coli

Quote:
Originally Posted by Jorge1907 View Post
What is the protein and why should its synthesis parallel the growth curve?
I'm working with an integral membrane protein (algal cycloxygenase like enzyme) Mw 67 kD. I must admit I am no expert in microbiology, but I have understood that E.Coli gives optimal yields when induced during logarithmic growth phase.
Reply With Quote
  #4  
Old 02-23-2009, 10:07 PM
Ph.D Doctorate
Points: 3,253, Level: 37 Points: 3,253, Level: 37 Points: 3,253, Level: 37
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2008
Posts: 154
Thanks: 0
Thanked 17 Times in 16 Posts
Default Re: Trying to explain odd effect observed in recombinant E.Coli

Maybe you've disproven that understanding. Have you reproduced the phenomenon?
Reply With Quote
  #5  
Old 02-23-2009, 10:29 PM
Aga's Avatar
Aga Aga is offline
Post-Doc
Points: 2,239, Level: 30 Points: 2,239, Level: 30 Points: 2,239, Level: 30
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 270
Thanks: 25
Thanked 78 Times in 64 Posts
Default Re: Trying to explain odd effect observed in recombinant E.Coli

Jorge may be right. That could be actually very interesting. Some proteins are expressed late, during stationary phase.

Try to be critical in everything you do and read - it pays off.
Reply With Quote
  #6  
Old 07-08-2009, 08:22 PM
Pipette Filler
Points: 56, Level: 1 Points: 56, Level: 1 Points: 56, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 9
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: Trying to explain odd effect observed in recombinant E.Coli

When looking at lots of recombinant proteins this is a common phenomenon. I was once told it was down to E. coli stationary phase sigma factors recognising the T7 promoter. 30 % of the proteins I produce, on average, express better when we "stew" them over-night un-induced. You should always do all the options though!

Good-luck!
Reply With Quote
Reply

Tags
ecoli , effect , explain , observed , odd , recombinant


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
FFiMP: Misconceptions about Special Relativity Jan Gooral Physics Forum 0 05-22-2008 02:53 PM
FFiMP: Spacetime Curvature and Gravitation Jan Gooral Physics Forum 0 05-22-2008 02:52 PM
What is Gravity? sdr@sdrodrian.com Physics Forum 1 01-27-2008 02:53 PM
The Achilles Heel of String Theory. S D Rodrian Physics Forum 7 07-08-2006 02:40 PM


All times are GMT. The time now is 07:26 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.17781 seconds with 16 queries