Electroporation with a pool of vectors with different inserts - why only one kind of vector maintained in the cells? (Jul/07/2007 )
I have made electroporation with a pool of vectors with different inserts to create a library. That have been made succesfully and I have clones with different inserts. But I wonder about one thing: What is the mechanism for the clones to maintain ONLY ONE kind of vector (insert) in the cell...??? When they have been bombarded with different kinds and they all have the same antibiotic selection gene.. competition??
i think it's more because the enter of a vector in a cell is quite a rare event. hence, the event of 2molecules entering a single cell may be so rare that it's usually not happening.
I take it that you are talking about transformation into bacteria to make library. If so, the ability to maintain 1 plasmid (or rather a number of the same copies of one plasmid) per cell is due to plasmid incompatability. Two plasmids are considered incompatable if they are unable to co-exist stably. This is because they share the features that regulate their replication, which means the replicator region, which contains the ori and all the sequences that encoding anything that participates in the replication of plasmid, e.g. RNAII, RNAI...
When you make library, the pool of inserts that you use is normally constructed using the same type of vector, so that all the vectors in the pool have the same backbone, the same ori region, eventhough they got diffrent inserts. So they are all incompatible. When they are transformed into bacteria, eventhough more than one vector could go into each individual bacteria, after the selection, growing of bacteria... the many incompatible plasmids in each cell will be lost randomly, until at the stable state, one bacteria will carry copies of only one stable plasmid.