NEW HOST-VECTOR SYSTEM ALLOWING SELECTION FOR FOREIGN DNA INSERTS IN BACTERIOPHAGE-LAMBDA GTWES

An improved vector (λgtWES.T5-622) for EcoRI fragments has been derived from EK2 vector λgtWES.λB' by replacing the λB fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parentaltype recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into λgtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Escherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the λgt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the λ DNA. © 1979.