## 1 Introduction  
The mechanism for bi-directional initiation of bacteriophage \(\lambda\) DNA replication involves a complex interaction of \(\lambda\) proteins O and P with the _E. coli_ host DNA replication machinery. The arrangement of genes \(O\) and \(P\) on the \(\lambda\) genome is shown in Figure 1. Their expression depends upon _pR-cro-cII-O-P_ mRNA synthesis from promoter _pR_ which is negatively regulated by the CI repressor, made from gene _cI_, binding to _oR_ operator sequences overlapping _pR_. The gp\(O\) (= O) acts to bind _ori\(\lambda\)_, the \(\lambda\) origin of replication, situated midway within the \(O\) sequence [1]. The gp\(P\) = P protein facilitates replication initiation through recruitment of an _E. coli_ host protein(s) to form a preprimosomal initiation complex at _ori\(\lambda\)_. In agreement with earlier genetic studies, P was found to physically interact [2] with DnaB the major replicative helicase for unwinding double-stranded (ds) DNA. DnaB promotes the advancement of a growing replication fork [3] by using energy provided by ATP hydrolysis; it functions as a \"mobile promoter\" in the general priming reaction [4, 5] to aid DnaG primase in producing RNA primers for extension by DNA polymerase III; and it is able to promote progression of Holliday Junctions in the repair of DNA damage [6, 7]. DnaB is a hexameric homomultimer with up to six bound ATP molecules [8, 9]. Its intrinsic ssDNA binding activity is inhibited when the free form of DnaB forms a complex with the host replication initiation protein DnaC ([10] and included references). P can commandeer DnaB away from DnaC [11]. P:DnaB participates in loading DnaB onto DNA during formation of the DnaB:P:O:_ori\(\lambda\)_ preprimosomal complex. The interaction between P and DnaB inactivates the helicase activity of DnaB. Restoration of DnaB activity, coupled with activation of _ori\(\lambda\)_-dependent replication initiation involves the dissociation of P bound to DnaB by _E. coli_ heat shock proteins DnaK, DnaJ, and GrpE [12, 13]. The ability of P to dissociate preformed cellular DnaB-DnaC complexes, coupled with a low cellular concentration of DnaB (\(\sim\)20 hexamers/cell) [14, 15], suggests the hypothesis that \(\lambda\)_P_ expression can inhibit _E. coli_ DNA replication initiation from _oriC_, alluded to herein as P-lethality. Figure 1: Relevant \(\lambda\) genes in expression plasmids and prophage. (**A**) Arrangement of synthetic plasmid pclpR-[GOI]-timm. Plasmids employed include the precise coding sequence for GOI (gene of interest) genes _O, P,_ or alleles of \(P\), including _P_\({}^{\pi}\), _P_-SPA, _P_\({}^{\Lambda 76}\), each inserted, in precisely the same orientation and position as gene _cro_ in \(\lambda\), directly downstream of _pR_. The coding sequence for the GOI is terminated by an ochre stop codon inserted just ahead of the powerful _timm_ termination sequence [16, 17], previously named _ti_[18, 19]. The regulatory regions of pclpR-(GOI)-timm were described (Figure 1 plus supplemental sequence file in [20]). The expression of the GOI from promoter _pR_ is negatively regulated by the lambda CI[Ts] repressor binding to the wild type _oR_ operator sites overlapping _pR_. These plasmids do not contain the _oL_ operator sequences so that the tightest repression of transcription from the _pR_ promoter, requiring CI-mediated DNA looping via CI dimers binding both operator sites [21], is not possible. Transcriptional read through beyond the GOI is prevented by _timm_, shown in its natural map position between the left operators, _oL_ and the C-terminal end of _rexB_ (see gene map drawn in Figure 1B). The synthetic sequence from the translational termination sequence for GOI through _timm_ to downstream _EcoR_I-_Sal_ is: _TAATCGAT_cccggGG_tcagCCccgggtttttt_TGAATTCGTGCGC_, where the bases that were modified from the wild type lambda DNA sequence are in capitalized italics.