A Phage Vector for Bacteria

A Phage Vector for Bacteria

Phage vectors and phage-derived vectors are useful for three reasons. Phage can carry larger inserted DNA fragments than plasmids. There-fore substantially fewer transformed candidates must be examined to find a desired clone. The efficiency of infecting repackaged phage DNA into cells is considerably greater than the efficiency of transforming plasmid DNA into cells. This is an important factor when a rare clone is being sought. Finally, lambda phage permits a convenient method for screening to detect the clone carrying the desired gene. Once a desired segment of DNA has been cloned on a phage, however, the convenience of manipulating plasmids, due in part to their smaller size, dictates that the segment be subcloned to a plasmid.

Lambda was an ideal choice for a phage vector because it is well understood and easy to work with. Most importantly, the phage contains a sizable nonessential internal region flanked by EcoRI cleavage sites. Therefore this nonessential region could be removed and foreign DNA inserted. Before EcoRI-cleaved lambda DNA could be used for cloning, it was necessary to eliminate additional cleavage sites that are located in essential regions of the lambda genome. First, a lambda hybrid phage

was constructed by in vivo genetic recombination. This lacked the three EcoRI cleavage sites at 0.438, 0.538, and 0.654 in the nonessential regionbut retained the two sites in the essential regions. Then the two remain-ing EcoRI cleavage sites were eliminated by mutation and selection. In the selection the phage were cycled between hosts lacking and contain-ing the EcoRI restriction-modification systems. Any phage with cleav-age sites mutated so as to be nonrecognizable by the EcoRI system would have a greater probability of escaping the restriction enzymes upon growth in the second host. After 10 to 20 cycles of this selection scheme, Davis found a phage that had lost one of the two R1 sites, and after an additional 9 to 10 cycles, he found a mutant that had lost the other site. This mutant phage was then made into a useful cloning vector by recombination with wild-type lambda to restore the three EcoRI cleavage sites located in the central portion of the genome.

The DNA isolated from lambda phage particles is linear and it can be cleaved by EcoRI to yield the smaller central fragments and the larger left and right arms. EcoRI-cleaved DNA fragments to be cloned can then be hybridized together and ligated to purified right and left arms. This DNA either can be used as it is to transfect cells made competent for its uptake or it can be packaged in vitro into phage heads and used to infect cells. The efficiency, per DNA molecule, of packaging and infection is much higher than transfecting with bare DNA. Therefore packaging is used when the fragment to be cloned is present in only a few copies.