Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Diauxic growth after IPTG induction

IPTG growth arrest

  • Please log in to reply
No replies to this topic

#1 Amro

Amro

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 03 October 2014 - 08:06 AM

Hi,

I'm working with a protein that I'm expressing in E. coli with an IPTG inducible plasmid. The protein is eukaryotic and I've checked it for rare codons (19 codons under treshold out of 93 but they're mostly taken care of by pRARE). I've also checked that the construct is in-frame, correctly oriented and that no mutations have occured. I checked if there is any leaky expression (none visible) and whether the protein really is produced and where it ends up (pellet or supernatant).

 

The thing is, about 30 min to 1h after induction the OD enters kind of a stationary phase (sometimes the OD even decreases a bit) where it remains for about two hours before it starts growing again. An uninduced culture will grow normally while a culture with added 1% glucose at the time of induction will grow normally for an hour until it enters a stationary phase as well. This is followed by a new growth phase.

 

The protein is a cytolytic toxin, with hydrophobic domains, that can lyse erythrocytes with a detergent mode of action. From what I´ve learned about the expression of this protein in E. coli, it ends up in inclusion bodies, but I've yet to isolate it in pure form.

 

I'm trying to understand if this growth arrest is due to the protein itself, if it has to do with the accumulation of inclusion bodies, or if the expression is responsible. Does anyone have a similar experience? What would the best way be to further study this phenomenon? Thanks.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.