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IPTG induction in non E.coli strains

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  #1  
Old 03-02-2007, 07:34 PM
Francesca
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Default IPTG induction in non E.coli strains



Hi...
I have a question, let me explain it:
vector pUC18 can be used as expression vector, as it has pLac and lacZ polylinker to clone gene of interest.
It is always suggested to clone in cells like E.coli JM109, XL1Blue etc. in order to perform the blue/white screening... ok?
But...
only for that? I mean. I knew that that strains were required for alfa-peptide formation and so IPTG/Xgal screening.
Are they also necessary to study IPTG induction?
IPTG binds to lac repressor coded from lacI, but pUC18 doesn't carry any lacI gene, so I suppose that lacI is coded from the host strains. Does it?
I've a plasmid, based on pUC18, and trying expression in a strain of Pseudomonas. IPTG induction doesn't work. Could it be because it lacks of lacI genes in its chromosome?

Thank you

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  #2  
Old 03-05-2007, 12:18 PM
Duncan Clark
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Default IPTG induction in non E.coli strains

Historians believe that in newspost
<[Only registered users see links. ].net > on Fri, 2 Mar 2007,
Francesca <[Only registered users see links. ]> penned the following literary masterpiece:

Yes. On an F'.


Never worked with Pseudomonas but several potential problems.

Will a Pseudomonas lacI bind to an E.coli lac operator and if so will
IPTG work in that system.

Will IPTG even get into Pseudomonas?

[Only registered users see links. ]

I take it you have searched Pubmed for any previous papers?

Duncan
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I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #3  
Old 03-19-2007, 11:19 AM
newsnet customer
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Default IPTG induction in non E.coli strains

>>IPTG binds to lac repressor coded from lacI, but pUC18 doesn't carry any

IPTG binds to the lac z operon to induce the lac z gene to express
beta-galactosidase. beta-galactosidase hydrolyses colorless X-gal to produce
insoluble blue preciptate (blue colonies). pUC18 does not have lacI gene as
this would inhibit the expression of beta-galactosidase, therefore blue
white screening would be impossible. For the same reason, the host strain is
a modified strain that also does contain the lacI gene.


There are papers on PUBMED that shows that blue white screening is possible
with Pseudomonas. I assume you did a positive control to check IPTG/Xgal is
working?

ST


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  #4  
Old 03-19-2007, 11:21 AM
newsnet customer
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Default IPTG induction in non E.coli strains

>>>IPTG binds to lac repressor coded from lacI, but pUC18 doesn't carry any


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  #5  
Old 03-19-2007, 06:05 PM
Kyle Legate
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Default IPTG induction in non E.coli strains

newsnet customer wrote:

Oh dear. It's a good thing for you you're anonymous. As you should have
learned in introductory biochemistry, lacI binds to the lac operon
(lacO), preventing transcription of LacZ-Y-A. IPTG binds lacI, which is
then released from lacO to permit transcription.
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  #6  
Old 03-20-2007, 10:52 AM
newsnet customer
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Default IPTG induction in non E.coli strains

>>>> IPTG binds to lac repressor coded from lacI, but pUC18 doesn't carry

lacI repressor protein binds to the lac operator and NOT the lac operon.

ST



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  #7  
Old 03-20-2007, 12:25 PM
Aawara Chowdhury
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Default IPTG induction in non E.coli strains

In <S1PLh.13817$[Only registered users see links. ].au>,
newsnet customer <[Only registered users see links. ]> wrote:


Since when did the lac operator stop being in the lac operon? And certainly
describing lacI to bind the lac operon is considerably more accurate than
your original description, "IPTG binds to the lac z operon .....".

AC
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  #8  
Old 03-20-2007, 09:33 PM
newsnet customer
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Default IPTG induction in non E.coli strains


"Aawara Chowdhury" <[Only registered users see links. ]> wrote in message
news:IoQLh.152241$[Only registered users see links. ]...

since it was first described.

ST


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  #9  
Old 03-21-2007, 02:28 AM
Aawara Chowdhury
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Default IPTG induction in non E.coli strains

In <qqYLh.13947$[Only registered users see links. ].au>,
newsnet customer <[Only registered users see links. ]> wrote:

No. Jacob and Monod described the operator as being part of the operon.
So you're wrong. But that isn't surprising, given your recent posting
history.

AC
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Email: echo 36434455860060025978157675027927670979097959886449 930P | dc
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  #10  
Old 03-21-2007, 08:00 AM
newsnet customer
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Default IPTG induction in non E.coli strains

>>> Since when did the lac operator stop being in the lac operon? And

No. The lac operator and the lac operon are two different entities. And if
your saying Jacob and Monod described it differently then you need to
provide a reference to support that statement otherwise its rubbish... just
like all your postings.

ST


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