3 replies to this topic

[Freemason]

Hello everyone,

I'm wondering if anything is known about cytotoxicity of GFP (alone, not as a fusion protein) when overexpressed in E.coli cells. Has anybody experienced some problems when transforming E.coli cells with a plasmid containing a T7 promoter and plating the transformants right afterwards on LB agar containing 1 mM IPTG? As far as I know, the concentration of IPTG doesn't matter that much when overexpressing proteins in E.coli, as there is only ON or OFF, and the expression level of the target protein isn't influenced by the concentration of IPTG itself (even if many papers suggest that).
Nevertheless, I would like to hear if somebody has ever observed something like that when working with GFP.

Thanks in advance,

Freemason

[protolder]

Hola, Today in the protein and proteomic forum, Mica asks for problems expressing GFP in E.coli. Buen día

[Freemason]

protolder, on 08 February 2011 - 11:15 PM, said:

Hola, Today in the protein and proteomic forum, Mica asks for problems expressing GFP in E.coli. Buen día

Thanks for the hint!

Greetings
Freemason

[pDNA]

From my point of view every protein expressed with the T7 System is somehow toxic to the cell ...since massiv amounts of mRNA are generated by the T7-system and therefore the translation machinery is titrated from essential cellular mRNAs. Therefore, most of the time induction with IPTG leads to cessation of growth of the culture.

Your statement that the concentration of IPTG does not matter is false ...it does matter ...but it is heavily dependent on the plasmid you use and the host background ...don't assume that a biological system is not able to sense concentration of an inductor or a carbon source ...it does

What leads you to your suspicion that GFP might be toxic to the cells ...maybe you can explain that in detail?

Regards,
p

Freemason, on 08 February 2011 - 08:50 AM, said:

Hello everyone,

I'm wondering if anything is known about cytotoxicity of GFP (alone, not as a fusion protein) when overexpressed in E.coli cells. Has anybody experienced some problems when transforming E.coli cells with a plasmid containing a T7 promoter and plating the transformants right afterwards on LB agar containing 1 mM IPTG? As far as I know, the concentration of IPTG doesn't matter that much when overexpressing proteins in E.coli, as there is only ON or OFF, and the expression level of the target protein isn't influenced by the concentration of IPTG itself (even if many papers suggest that).
Nevertheless, I would like to hear if somebody has ever observed something like that when working with GFP.

Thanks in advance,

Freemason