Protein expression from bacteria which has two plasmids? - Incompatibility of plasmids (Jul/27/2007 )
I am planning to express two plasmids in bacteria. I will have two different pET vectors with F1 origin. They have diffrent antibiotic resistance. Has anybody had `bad`or `good`experience in expressing two pET vectors. I have read that it might be problematic to express two plasmids in bhttp://www.protocol-online.org/forums/style_images/1/folder_post_icons/icon2.gif
http://www.protocol-online.org/forums/styl...con2.gifacteria due to the incompatibility of two plasmids. But I could not find any info about the criteria to be considered as incompatible.
I would be grateful for your replies.
I dont really get it. Do you mean by transforming 2 plasmids inside the bacteria? Hmm.. this is the first time I heard about it. In my own opinion, I don't think it will be a very good idea to express 2 plasmids at one time. Expressing 1 plasmid itself is tough.
Thank you for your reply.
You got it right. Transforming two plasmids into bacteria. One postdoc in our lab told me that he even saw people transforming 5 plasmids into bacteria.
5 plasmids? Hmm what kind of bacteria can do so? I am just curious. Not an expert in it. =)
What you want to do is possible, as each plasmid type has its own antibiotic selection marker. However, the system is unstable as two plasmids from the same incompatability group interefere with the stable inheritance of each other. Thus the ratio of plasmid A to plasmid B will vary from cell to cell... some cells will have more plasmid A and others less. Which in turn results in the ratio of proteins expressed by each plasmid to differ between cells.
There are about 30 incompatability groups. So theoratically I could have ~30 plasmids living side by side, as long as each plasmid comes from a different incompatability group.
Still, 5 is a lot. The most I have read about is 4. The e coli cells weren't too happy. Too many antibiotics.
You could maintain 2 plasmids inside 1 E.coli, but you need:
- Different replicator factors (diffrent ori)
- Different selection markers.
Since you said that you have 2 diffrent pET vector with F1 origin, I take it that the ori region for both vectors is the same? If so, the two plasmids are incompatible, can't maintain for long in the same bacteria. Choose different ori.
Check this for more detailed explaination: http://www.protocol-online.org/forums/inde...showtopic=28736