## 3 Results  
### Thermally Inducible Gene Expression from pclpR[]Timm Plasmid  
In this report, plasmid-based synthetic \(clI\) constructs are transcribed from \(\lambda\) rightward promoter \(pR\), which is regulated by a \(cl\) [Ts] repressor binding to \(oR\), Figure 1 and which fully limits expression from \(pR\) at 30 \({}^{\circ}\)C. This plasmid does not include the \(oL\) operator so that octamer looping between the operators and CI repressor complex binding to produce a higher stability repression complex [42; 43; 44; 45] is not possible. Transcription-influencing factors (_cro_ expression, \(nutR\) and \(tR1\)) mapping between \(pR\) and \(clI\) were eliminated. This included removing an IHF (integration host factor) binding site (Figure 2) ahead of the RBS for \(clI\). This site was suggested both to stimulate \(clI\) transcription by suppressing transcriptional termination at \(tR1\)[46] and to enhance \(clI\) mRNA translation by preventing the formation of a RNA:RNA duplex that includes the RBS in the message ahead of \(clI\)[47]. The expression of a gene cloned downstream from the promoter \(pR\) within plasmid pclpR-[-]-timm is influenced by culture temperature, Table 1. Previously, cultures of 594[pclpR-GFP-timm] and 594[pclpR-D-GFP-timm] (where GFP was fused downstream from \(\lambda\) gene D) were used to show that both cell types formed fluorescent cfu (colony forming units) when grown up on spread plates incubated at 37, 39 or 42 \({}^{\circ}\)C, compared to colonies grown up at 30 \({}^{\circ}\)C which did not fluoresce [29]. In these GFP plasmids the RBS for _cro_ was positioned as described in Figure 1 for [clI], as drawn in the middle structure, Figure 2. Table 1 shows the kinetics of D-GFP fusion protein expression from \(pR\) upon shifting the culture temperature of 594[pclpR-D-GFP-timm] cells growing at 30 \({}^{\circ}\)C to 37,39 or 42 \({}^{\circ}\)C. GFP expression in cells raised to 37 \({}^{\circ}\)C was not observed until about 60-120 min post induction. The level of gene expression for cells raised to 42 \({}^{\circ}\)C by 150-180 min post induction was 43-75X that over cells raised to 37 \({}^{\circ}\)C and 8-10-fold over cells raised to 39 \({}^{\circ}\)C. By analogy, it is assumed that when _clI_ is cloned downstream from promoter _pR_, its expression will resemble that of the D-GFP fusion protein. The ability of the Ts CI857 repressor encoded by the plasmid to reduce transcription from _pR_ at 37 \({}^{\circ}\)C is lost at 42 \({}^{\circ}\)C. Limited _pR_ gene expression at both 36 and 37 \({}^{\circ}\)C was previously observed in studies where the gene for the highly toxic P protein of \(\lambda\) was cloned downstream of _pR_ in pclpR-P-timm [38]. Figure 2: Potential stem-loop configurations in _pR_ mRNA ahead of _clI_ drawn from \(\lambda\)WT and synthetic _clI_ expression plasmids. In each structure, the start site for _pR_-initiated mRNA is suggested as arising at \(\lambda\) sequence base 38,302 just downstream from _pR_[48]. The structure on left omits the _cro-nutR_-_tR1_ sequence and was inspired from a drawing in [36] showing the potential for stem loop mRNA secondary structure ahead of the translational start for _clI_ that could impede translational initiation. The FMet codon for _clI_ is green. An IHF (integration host factor) binding site in DNA [47] is shown in red which resembles the IHF consensus sequence AAAAAAnnTTnnnWATCAAnnnTTR (where W is A or T, R is A or G and N is any nucleotide) but lacks the A-tract region about one helical turn from the consensus region [49]. The positions for some of the mutations [36] influencing _clI_ that fall within the stem loop are shown. The structure in the middle includes the mRNA sequence produced from plasmid [CII]. This eliminates the stem-loop mRNA structure ahead of _clI_ found in WT \(\lambda\).