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. 2018 Mar 7;10(3):115. doi: 10.3390/v10030115

Figure 1.

Figure 1

λ imm-region map and synthetic cII plasmid expression vector. (A) Map of the imm-O region of λ showing immunity region genes rexB—cro, several promoters (those relevant are shown in orange) and terminators, tImm, tR1 and tO. The cI maintenance promoter, pM enables the expression of gene cI, encoding a repressor that binds to operators oL and oR and blocks transcription initiation from the major leftward and rightward promoters pL and pR. pO is required for transcription of the short OOP RNA [21]. The product of gene cII, transcribed from pR, stimulates repressor establishment transcription from promoter pE. The level of pE transcription can be 30- to 100-fold greater than the level of cI transcription from promoter pM upon thermal induction of a cro defective prophage [22,23,24] or 10- to 20-fold after λ infection [25]. tImm, terminates repressor establishment transcription from promoters pM and pE, preventing read-through into the oL/pR site. (B) Plasmid pcIpR-cII-timm (abbreviated herein as [cII] in tables) is a synthetic cII expression system (see Table S1 for related constructs) that eliminates gene cro, ninR and the tR1 terminator, as shown in the plasmid insert (not drawn to scale). Gene expression from the pR promoter is regulated by the encoded temperature sensitive CI [Ts857] repressor, via binding to the oR operator sequences. Gene cII is positioned immediately downstream of the WT ribosomal binding site (RBS) for cro, which perfectly matches/can base pair with the 3′ terminal AUUCCUCCA sequence of 16S rRNA [26]. Potential stem-loop configurations in pR mRNA ahead of cII for the various plasmid constructs used are shown in Figure 2. Sequence variations (1–3) of pcIpR-cII-timm enabling cII expression arrangements are shown in Figure S1. The overall organization of genetic elements on plasmid pcIpR-[…]-timm was described [27,28,29], where a synthetic version of tImm is inserted between the ClaI and EcoRI sites to prevent read-through of transcription arising from promoter pR.