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
- - - - -

pET47b proteins not expressing in BL21 (DE3)


  • Please log in to reply
5 replies to this topic

#1 Luria Bertani

Luria Bertani

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 56 posts
0
Neutral

Posted 22 August 2011 - 10:06 PM

Hi there,

I have a gene for a 25 kDa bacterial metallo protein placed into pET47b / BL21 (DE3). I've done an expression trial and I can't see any bands for expression-- what are some of the options I can do to play around with to see if it can express?

#2 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 493 posts
14
Good

Posted 23 August 2011 - 11:49 AM

have you checked the construct if everything is in frame and free of mutations prior to expression?

if you want help you have to give more details on your expression experiment e.g.
- IPTG concentration used
- duration of expression after induction
- Optical density at induction
- course of OD after induction (growth/no growth)
- confirmation expression (PAGE, western blot, ...?)

if you give us more details we can give you suggestions!

Regards,
p

#3 Luria Bertani

Luria Bertani

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 56 posts
0
Neutral

Posted 23 August 2011 - 08:44 PM

Hi pDNA, thanks for your reply.

This was an expression test, the construct itself seems fine and has been sequenced. The gene is under T7 promoter control.
The IPTG concentrations used were 0mM, 0.1 mM, 0.5 mM, 1 mM
Induction took place when the OD600 reached 0.5 (I didn't measure the OD after induction)
The duration of the expression was 24 hours with samples taken at 3 hours, 6 hours, 12 hours and 24 hours after induction
SDS-PAGE was used to check for any bands that might appear- none seem to jump out

#4 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 23 August 2011 - 10:30 PM

Hola, As always I say I think that the problem is the host.It hasn´t any control to repress any basal expression which could give the extinction of producers recombinant bacteria at first stages of culture , surviving no producers . This is noticed when early growth phases are too long.
Whit this host I would change tryptone by soytone, and add a low glucose (o.1-0.2 g/l) concentration from the seed. If the problem isn´t solved I woud change to any LacIq strain, or better to any lac- lacIq strain as Origami (DE3 lacI) or better OrigamiB DE3 placI , LacI of the plasmid isn´t enought to repress early expression. Cross your fingers and buena suerte

#5 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 493 posts
14
Good

Posted 24 August 2011 - 11:13 AM

what antibiotic? ...Ampicillin?

Regards,
p

#6 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 24 August 2011 - 09:38 PM

Hola Peter Sorry by my bad expression, I mean add glucose from the preinoculum. Not always glucose repress expression but sometimes it helps (uv5 promoter isn´t sensible at glucose). Other possibility is using as preinoculum all the colonies of a fresh transformation whit pure plasmid and follow the expression from the begining withouth induction and induce with lactose o0.5 g/l. buena suerte




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

©1999-2013 Protocol Online, All rights reserved.