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Does over-expression protein under T7 promoter in Top10 work?

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#1 paulomach



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Posted 15 December 2015 - 11:41 PM

In my lab, someone does the over-expression of GFP under a T7 promoter (pRSET vector) into Top10 E.coli strain and it works!

I don't understand how it works without DE3 in E.coli's genome?

Do you have an explaination?

Thanks :-)



#2 labtastic



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Posted 16 December 2015 - 06:44 AM

What you are probably seeing is leaky expression, which is not uncommon with T7-based expression systems. They are probably getting only a fraction of the GFP they could be getting.


To get full expression you need to have a strain of Ecoli that has the T7 polymerase encoded with in its genome. The T7 polymerase is what drives transcription of your gene, which is under the control of a T7 promoter.


The DE3 part of BL21(DE3) means the Ecoli has this T7 polymerase inserted into the genome via phage DNA.


There are other commercially available strains of Ecoli that also have T7 polymerase encoded in their genome, but not via phage DNA but via more modern molecular biology methods. Examples include BL21ai and NEB's T7 express cells both of which work quite nicely.


However, you can express pBad based plasmids (i.e. arabinose inducible) in TOP10 cells just as well as any other system.

Edited by labtastic, 16 December 2015 - 07:06 AM.

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