May I know what is the maximum number of plasmids (with different origins of replication) can be taken up by a cell? What factors affect this? The maximum I have tried was two.
I think by artificial transformation (electroporation or CaCl2 method, etc.) only one plasmid can enter the cell. Even if two plasmids with different origin of replication exists within the same cell, the copy number of each plasmid and the copy number control of them would determine the chances of their co-existence. That would determine the compatibility of those plasmids. If the plasmids have same or similar origin then they can control each other's replication and that might cure the cell of one of the plasmids. If they have completely different origin, then the replication of one should not affect the other and both independently replicate and co-exist. Also, some plasmids may influence the partitioning (the division of plasmids between 2 daughter cells) and may result in unequal or biased inheritance of plasmids in the daughter cells. Like one may inherit higher number of plasmid A and lower number of plasmid B. So I guess, replication of plasmids (copy number control mechanism) and partitioning effects are the major factors to determine plasmid compatibility (whether or not they can co-exist in same host).
Dear Teng Tai,
Number of plasmids (whether of same or different origin of replication) taken up simultaneously by the bacteria pretty much depends on transformation efficiency of the bacteria.
Regarding plasmid incompatibility, I came across a very interesting paper entitled "Plasmid incompatibility: more compatible than previously thought?". You may want to go through it.
http://peds.oxfordjournals.org/content/20/7/309.long
I have another question here. They said that plasmids with same origin of replication cannot coexist. So when we do transformation, only one plasmid will enter one cell at one time. The question is, after we clone the plasmid in, they will replicate inside the cell. So at this point, a cell will contain many plasmids with same origin of replication inside its body. Why these plasmids can coexist and no rejection occurs?
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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