Ampicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it serves it’s purpose, there can be problems using this selection marker if the user is unaware of it’s limitations. This article provides a quick overview of what these limitations are and how to avoid them.
The basis of ampicillin selection is the hydrolysis and inactivation of the antibiotic by beta-lactamase expressed from the plasmid-borne bla gene. Here’s the problem: beta-lactamase is secreted by the bacteria. The resulting build-up of extracellular beta-lactamase can inactivate the ampicillin in the culture medium, removing the selective pressure, if the culture is not handled properly.
In liquid cultures, this means that a portion (possibly a very large portion) of the cells no longer have the plasmid, giving poor yielding plasmid preps, protein expression etc. On agar plates, ampicillin degradation can lead to the formation of satellite colonies on transformation plates. Satellite colonies are very small colonies of cells that have not taken up the plasmid that form around a large colony that has taken up the bla-containing plasmid. The satellites form because the beta-lactamase released by the bla-expressing colony degrades the ampicillin in the vicinity of the colony. The satellites are not necessarily a problem as they will not grow when transferred to a medium containing fresh ampicillin.
So how do you avoid plasmid loss when using ampicillin as a selection marker? Here’s how:
Do you have any tips on working with ampicillin? If so, please leave a comment.