Transformation of a plasmid into E.coli BL21 - problem! - Unexpected bands (Sep/23/2006 )
Hello friends, I like this page a lot for useful comments. Now I have a serious problem. I purified a plasmid, which I had amplified in E.coli omni strain. I load the purified plasmid (4µl) on a gel and there was one band - the plasmid, everthing is great. Then I did the transformation of this purified plasmid into the E.coli strain BL21 to express the protein, for which the plasmid are coding for. Omni strain has not the translational machinery to express the protein. Before doing the protein expression, I checked if the E.coli cells have taken the plasmid inside the cell. I purified the plasmid out of that cells like I did in E.coli omni strain and load the purified plasmid on a gel (4µl) and there was not only the plasmid but also four other bands above this plasmid. I thought it can be a DNA contamination, so I did the gel extraction: I load the hole amount of my plasmid 30µl on a gel and purified just the single band from my plasmid and load 4µl again on a gel. But there were also these bands! I got crazy! What can that be. I have recognized these bands just in E.coli BL21 not in omni strain. A friend says there are supercoiled forms and nicked forms of the plasmid, but the molecular weight was at 10.000 to 6000kDA and my plasmid has about 3000kDa, so it cannot be another modification of my plasmid. Had anyone similar experiences or even an idea? Thanks a lot for good comments, because my boss hates me...
The other bands are most likely other forms of the plasmid. To check it, cut a small mount of the plasmid prep with an enzyme that cuts once, and run that out on a gel. If you see a single band (assuming complete digestion), there ya go...
A linear molecular weight marker tells you nothing about the actual sizes of circular DNA, it's an invalid comparison.
Homebrew is right...these bands could be other forms of your plasmid, different plasmid conformations migrate through the gel in different ways
Thanks a lot for your replies! So why are these four bands not in E.coli omni strain? Why do they appear just in E.coli BL21?
every DNA prep is different; it is not necessarily a difference between strains.
on a sample of uncut plasmid DNA, you can see anywhere from 1 to 6 bands that are all the same plasmid in different forms. some times it may be due to differences between one plasmid and the next (I'm guessing dimerization will occur more easily with some plasmids and others, secondary structure and the like?) but things like nicked forms and such have to do mostly with the prepping protocol
My suggestion would be to linearise you plasmid with at least 2 different enzymes. If the problem goes away, then everything should be okay. Also cut the plasmid into fragments and see if the gross structure is okay.
There is one last card that can be played if your observations are real (linear DNA also show multibands).... recombination
Unless it is specially mentioned by the person who gave you the BL21 cells, it should be noted that most BL21 derivitive strains are recA+. BL21 will recombine your plasmid, and may produce larger plasmids.
If you are doing your cloning work in BL21, I would strongly suggest a different strain be used. recA+ cells are not something you want to handle during long cloning steps.
ok I'm going to try to cut it with two enzymes and let's see what will happen! What does it means if a strain is recA+? I've never heard about that.
RecA is an endorgenous ssDNA binding protein of E. coli. It plays a critical role in homologous recombination (also a part of DNA repair). RecA helps the initiate ssDNA strand invasion of dsDNA and it is RecA which causes the further unwinding that allows branch migration.
Homologous recombination (or any recombination) is bad news for the plasmid.
Nearly all cloning strains have the recA gene knocked out (recA-). However some (many?) protein expression strains retain the recA as a functional gene because recA mutants grow much more slowly.