how many plasmids can a competent bacteria take - (Aug/22/2008 )
i would appreciate if any body can tell me how many plasmids a competent bacteria can take.
i am actually transforming 2 plasmids which differ only in a tag and i cant separate them before transformation bcz i myself add the tag. so i just want to know if competent bacteria take only one plasmid or they can take several, if the first one is true i can simply separat them with colony picking. otherwise gel purification and dna sequencing must come to help me work it out
If you asking ... if I have 2 different plasmids in my mix, will both be taken up in the same bug? The answer is YES.
what is your tag ... if it is a genetic tag, you could use PCR to identify which clone contains only the tagged-plasmid.
Hope this helps,
I was been taught that it is very low chance for one competent cell to take more than 2 different plasmids.
I also believe that this is a rare problem. Even if it were common, you can achieve your desired result by transforming with serial dilutions of your ligation, and picking colonies from the more dilute transformation plates.
I hope not! Our planned system, using the pETDuet family, will need up to 5 plasmids...
to my knowledge transformation doesnot depends on kind of plasmid. any kind of plasmid can be inserted into bacteria. its a physical process.
There's a difference between putting all of the plasmids into a cell at the same time and putting them in one at a time. For inserting five, you would do them one at a time with selection at each stage. I thought the question concerned the insertion of more than one plasmid from solution in the same transformation round.
The plasmids must be of different incompatibility groups to stably coexist in the same cell...
many thanks to your replies,
both plasmids are pcDNA 3.1(+), the insert is also the same but there is a flag tag only in one of them.
i am transforming them at the same time and i cant separate them before transformation.
i cant get what u mean by "The plasmids must be of different incompatibility groups to stably coexist in the same cell"
Say we call one plasmid "A" and the other "B". At the time of transformation, you may have a single cell with both "A" and "B" resident. However, it is unlikely that this situation is what you need; rather, I'm guessing you probably want this cell to divide, and for each of the daughter cells to also contain "A" and "B", and for these daughter cells to divide and each of their siblings to contain "A" and "B", etc., etc., and for each of the plasmids to express the genes they carry.
A problem with this scenario arises if plasmids "A" and "B" belong to the same incompatibility (Inc) group. Plasmids do not exist singly in a cell, but have a genetically determined copy number they strive to maintain. Plasmids belonging to the same incompatibility group interfere with the stable segregation of each other into progeny cells so that, over time, one or the other plasmid is disproportionately represented in any given cell; many times one or the other plasmid is completely displaced and thus lost in the population.
For more information, see the "Incompatibility Groups" section here, see the sections on "Incompatibility testing" and "Incompatibility groups" in the Google Books preview of Plasmid Biology by Gregory Phillips, Barbara E. Funnell here, or Google "plasmid incompatibility groups".