Investigating Toxicity of Bacteriophage Lambda N-Protein Upon Overexpression in <i>Escherichia coli</i>

Abstract

Over the course of two decades, cloning of a variety of native and engineered DNA fragments from bacteriophage lambda was performed as part of a capstone undergraduate course aimed at teaching fundamentals of recombinant DNA technology and regulation of gene expression in bacteria. Genomic DNA of bacteriophage lambda (λ–phage) was digested with BamHI and HindIII and the resulting DNA fragments were then ligated into similarly digested plasmid vector pUC19 to illustrate the principle of shot-gun cloning. E. coli strain TB1 was then transformed using selection for ampicillin resistance and the blue-white color screen. Upon analysis of recombinant plasmids isolated from white colonies, it became apparent that four of the five BamHI and HindIII DNA fragments were easily obtained, but one fragment was persistently missing. This 2396 bp BamHI-HindIII fragment between lambda phage genome coordinates 34500-36895 (Accession No. J02459.1) was dubbed the toxic fragment and contained the strong leftward promoter (pL) and downstream N gene encoding the transcription anti-terminator protein N, as well as the ‘rexA rexB genes. Truncation of this toxic fragment revealed that a smaller fragment (1,133 bp, spanning coordinates 34500-35632) was sufficient to confer toxicity upon cloning into pUC19. This smaller fragment contains the intact pL promoter, the open reading frame for the N-protein, and, importantly, 540 bp of 3’ untranslated DNA (UTR, which contains transcription terminator tL1). High level expression of the N-protein was hypothesized to be responsible for bacterial toxicity. This hypothesis was validated when the shorter (1,133 bp) toxic fragment was successfully transformed into strain TB1 containing pACλcI encoding lambda phage repressor protein which negatively controls expression of the N protein by binding to operators within the pL promoter. In addition, various mutations altering the pL promoter relieved toxicity. To further narrow the region of the UTR and/or N-protein responsible for bacterial toxicity, a series of truncations was created by PCR. Surprisingly, a recombinant plasmid containing the wild-type pL promoter and full length N-gene (but lacking tL1) was not toxic. Current research is focused on truncating and mutating bases within this 3’-UTR with the goal of understanding what role tL1 may be playing in toxicity