Novel lambdoid cloning vectors.

The design of the general-purpose lambda cloning vector is analysed to determine several specific areas of usefulness and ideas are developed to improve the usefulness of lambda vectors in four of these areas: ease of use, cloning capacity, selection of recombinants and recombinational stability of cloned sequences. To make a vector which is easier to use, a derivative of EMBL4 which can be used to make recombinant phage capable of forming lysogens in a special host is constructed and tested to determine if high titre phage lysates can be prepared by thermo-induction of such a lysogen. To increase the cloning capacity of lambda vectors a special host lysogen capable of providing all the lambda late gene products is constructed and tested. Growth on this special lysogen allows the replaceable region of the phage vector to be extended through the left arm to the B gene giving a vector with 35 kilobases of capacity. To improve the selection of recombinant phage, a novel central fragment counterselection using the Tro phenotype, and which will work in the absence of host recombination systems, is designed and tested. During investigations of recombination-deficient host strains a novel, rec-independent recombination system is defined in E. coli. .