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Co-expression of 2 plasmids in E.coli


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#1 Chris Kar

Chris Kar

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Posted 03 April 2012 - 05:36 AM

Hi!

I am trying to develop a screening system using E.coli and therefore, I have to co - express 2 plasmids in a single E.coli cell. I have generated totally from the beginning 2 plasmids using components from other plasmids; that means I have constructed the following plasmids:

1. for my gene of interest, I have a vector with Kanamycin resistance, tet constitutive promoter and CDF origin of replication.

2. the second plasmid I constructed, contains Ampicillin resistance, lac promoter and pBR322 origin of replication; in principle this is the pUC8 vector.

In plasmid No 2, I have cloned eGFP, because this will be my inducible expression gene.

When I am expressing the eGFP alone using either lactose or IPTG, the protein production is perfect; my problems start when I transform the cells harboring already the pUC8-eGFP, with the plasmid No 1 for co-expression. In the last case the eGFP is not produced and somehow is repressed!

I have performed ALL the antibiotic resistance controls and they really work as well as all constructs are sequenced.

But simply, the pUC8eGFP is not working in terms of expression, when the other plasmid is present; I underline the fact, that the pUC8eGFP is maintained by the cells because I have both different origin of replication and antibiotic markers. My problem is the expression.


Any ideas, suggestions are extremely welcome.......

Thanks a lot in advanced,

Best,
Chris

#2 Laforet

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Posted 05 April 2012 - 01:30 PM

Lac promoter is rather weak and people generally don't use pUC for protein expression for the dry same reason. Clone your egfp into a different vector with a stronger promoter such as tac might make it work.

Otherwise, try growing up cells in media with 1% lactose as the only carbon source.




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