Figure 1.

Relevant λ genes in expression plasmids and prophage. (A) Arrangement of synthetic plasmid pcIpR-[GOI]-timm. Plasmids employed include the precise coding sequence for GOI (gene of interest) genes O, P, or alleles of P, including P^π, P-SPA, P^Δ76, each inserted, in precisely the same orientation and position as gene cro in λ, 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 pcIpR-(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 EcoRI-SalI is: TAATCGATcccggGGtcagcCccgggttttctttTGAATTCGTCGAC, where the bases that were modified from the wild type lambda DNA sequence are in capitalized italics. Shifting cells with a pcIpR-[GOI]-timm plasmid that were grown at 25 or 30 ˚C, to above 39 ˚C induces expression of the GOI from the plasmid [20]. (B) Cryptic prophage in strain Y836. The λ phage genes cro-cII-O-P-ren are transcribed from promoter pR that is embedded within the rightward operator sequence, oR, between genes cI and cro. Gene cI, encoding a temperature sensitive repressor is transcribed from promoter pM; and cro is transcribed in the opposite direction from pR. C. At temperatures where CI remains active, i.e., at or below 38 ˚C, λ replication initiation is prevented, and the λ fragment is replicated as part of the E. coli chromosome by forks arising from oriC. At about 39 ˚C, CI becomes fully denatured, pR transcription is induced, and λ undergoes a few replication initiation events from oriλ [22].