Orientation dependent miniprep problem? - (Mar/10/2006 )

Hi all,

I did several searches, but i could'nt find a topic with the answer of my question. A few days ago i managed to clone a gene into a pUC plasmid containing a tac promoter. A did the ligation then I've transformed the construction into DH5a chemical competent cells. I used B/W selection with 0,1mM IPTG on LB Amp plates with X-Gal, and surprisingly I've got a plenty of white colonies (It was a blunt/blunt ligation with Klenowed fragments). I checked 20 of these white colonies with colony PCR in an orientation sensiteve manner, and I've fond that more than the halfo of my colonies contained the insert in a correct orientation. After this, I tried to do some minipreps on the positive clones and one negative control (white colony with no PCR product), and surprisingly i was only ble to purify plasmids from the colonies harboring the wrong ori pUC plasmid.

Interestingly I have cloned this gene from an other E. coli strain so it is impossible to be toxic for E coli. And the minipreps does not have any growth problems.

Mi explanation to this phenomena:
1, If the tac promoter is able to control the expression of my gene (it has'nt a toxic protein yield anyhow) the cells somehow reduce the copy number of the plasmid in order to survive some toxic effect of overproducing the coded protein (of course during minipreps I do not add IPTG to the culture).

2, The colony PCR gave me several false positive clones.

3, The high copy number of the pUC19 is too high to handle this gene.

Do you have any other idea to overcome this problem??

I don't have a solution, but maybe you can try growing them at a lower temperature like 18C.

I've once cloned a plasmid for a fusion-construct (in coli it was not in the expression vector), but somehow the coli's recognized a promotor-sequence and started to express it. There was GFP in it, so it was clear what they did. But the problem was that the transcription direction was opposite of the transcription direction of the ORI. So the plasmid survived a couple of hours in a miniculture, but after that, there was recombination and I could't find my plasmid back. Either they did not grow at all, or I got really lots and lots of DNA out of the miniprep, but not my construct but the empty vector.
But since this is an expression vector, I don't think that the transcription directions collide.