Transducing Phage Carrying Genes Other than gal and bio

Transducing Phage Carrying Genes Other than gal and bio

Isolation of gal or bio transducing phage is straightforward because these genes flank the lambda attachment site. For genes located farther away, a simple improper excision event cannot produce transducing phage. Genes located perhaps as far as a minute away on the genetic map, about 40,000 base pairs, from an integrated phage can be picked up by a combination of a deletion of the intervening DNA followed by an improper excision event. In this case the combined probabilities for such a double event are so low that large cell cultures must be used to find the transducing phage. What can be done to isolate phage that carry genes located more than a minute from attB of lambda?

Two approaches were used to bring an integrated phage and a particular gene close enough together to permit generation of transduc-ing phage. Either the gene was moved close to the integrated phage genome or the phage was moved close to the gene. The phage can be brought near the gene by selecting for lysogens following infection of a host deleted of the phage attachment region attB. At very low but usable frequencies, lambda will then integrate into sites that possess some similarity to the normal chromosomal integration site. These secondary integration sites are so widely scattered over the chromosome that lambda can be forced, albeit at very low frequencies, to integrate into or adjacent to most genes. Either the desired integrant can be selected by genetic means or the entire population of cells with phage integrated

at many different sites can be used as a source of phage. Some of the phage will excise incorrectly from their abnormal positions and transduce the desired adjacent bacterial genes. The second method of forcing an integrated phage to be near a target gene is to select for the illegitimate recombinational insertion of an episome carrying the gene into a site adjacent to the prophage.