In this design the luc gene is transcriptionally fused to xylS via overlapping stop and start codons and should be translated only when xylS is translated first. The new plasmid was designated as pFS7 (Figure 1). To test the functionality of this construct we used a series of xylS variant sequences which had been synthesized. These
variants contain synonymous codon changes relative to the wild type sequence and had been found to activate Pm to varying extents (in the presence of induction). We hypothesized that the effects of the codon changes were caused by variations in xylS mRNA translation, since transcript amounts CP673451 research buy were found to be similar to the levels of the wild type gene (qRT-PCR, data not shown). Nine such variant sequences were tested in pFS7, and luciferase activities were measured (Figure 2). The values varied in the range from about 20 to 100% of that of the construct containing the wild type xylS. Figure 1 Map of plasmid pFS7. Ps2: constitutive promoter; xylS: gene encoding Pm activator; luc: gene encoding luciferase; Pm: positively regulated promoter; bla: ampicillin Captisol cost resistance gene encoding β-lactamase; t 1 : rrnBT 1 T 2 bidirectional transcriptional
terminator; trfA: gene encoding the replication protein; oriV: origin of vegetative replication; kan: kanamycin resistance gene; oriT: origin of conjugal transfer. The DNA sequence of the overlapping stop-start codon is depicted. Figure 2 Expression levels from pFS7 for different variants of xylS with silent mutations. Relative expression levels from Pm (measured as maximum ampicillin tolerance at 1 mM m-toluate) are given in grey (error bars = lowest ampicillin concentrations
in test on which no growth was observed) and relative luciferase activity as a measure for XylS amounts in black Amisulpride (values from at least two biological replicas). All values (relative ampicillin tolerance and luciferase expression) refer to those of wild type XylS (Selleck JPH203 tolerating 350 μg mL-1), which are both arbitrarily set to 1. Mutations in the variants (1 to 9), the number stands for the base position that has been changed, relative to the translational start site, the character tells the base in the variant. 1: 6- > C; 2: 13- > C; 3: 15- > G; 4: 16- > C; 5: 27- > G; 6: 30- > C; 7: 36- > T; 8: 42- > T; 9: all of the eight mutations. The design of plasmid pFS7 also allowed us to study the effects of the changed XylS expression on activation of Pm. For this purpose the bla gene, encoding β-lactamase, was used as a reporter (see Figure 1). We have previously used this gene to monitor expression from Pm, since the tolerance of the host to ampicillin correlates well with the produced amounts of β-lactamase in a directly proportional way , up to ampicillin concentrations of 16 mg mL-1, thus making it easier to identify clones with desired phenotype without laborious library screening [10, 26, 27].