AN EXPRESSION-PACKAGING-PROCESSING VECTOR WHICH SELECTS AND MAINTAINS7-KB DNA INSERTS IN THE BLUE T4 PHAGE GENOME

We have developed an efficient positive-selection vector to insert for eign DNA segments fused to the T4 ipIII gene (encoding internal protei n IPIII) into the bacteriophage T4 genome. By using partial deletions of the T4 e gene, which encodes phage lysozyme, lysozyme activity requ ired for plaque formation is used to select plasmid integrants which r estore the e gene. In this work, we demonstrate that DNA inserts more than 7.0 kb in length can be incorporated into a T4 genome lacking the nit gene. In addition, the recombinant T4 not only contains a fusion gene driven by the T4 ipIII promoters, but also packages the fusion pr otein into the T4 capsid due to targeting by the IPIII portion. This e xpression-packaging-processing system shows that active IPIII::beta Ga l fusion reporter protein is produced and packaged during phage infect ion.