Two neglected but valuable genetic tools for Escherichia coli and other bacteria: In vivo cosmid packaging and inducible plasmid replication

In physiology and synthetic biology, it can be advantageous to introduce a gene into a naive bacterial host under conditions in which all cells receive the gene and remain fully functional. This cannot be done by the usual chemical transformation and electroporation methods due to low efficiency and cell death, respectively. However, in vivo packaging of plasmids (called cosmids) that contain the 223 bp cos site of phage λ results in phage particles that contain concatemers of the cosmid that can be transduced into all cells of a culture. An historical shortcoming of in vivo packaging of cosmids was inefficient packaging and contamination of the particles containing cosmid DNA with a great excess of infectious λ phage. Manipulation of the packaging phage and the host has eliminated these shortcomings resulting in particles that contain only cosmid DNA. Plasmids have the drawback that they can be difficult to remove from cells. Plasmids with conditional replication provide a means to “cure” plasmids from cells. The prevalent conditional replication plasmids are temperature‐sensitive plasmids, which are cured at high growth temperature. However, inducible replication plasmids are in some cases more useful, especially since this approach has been applied to plasmids having diverse replication and compatibility properties. Concatemers of a cosmid resulting from phage λ‐engendered rolling circle replication.