Ampicillin concentrations varied from 5 μg mL-1 to 4500 μg mL-1. Test of XylS expression levels using a synthetic operon and luciferase assay XylS amounts could be measured more directly Selleckchem Ferrostatin-1 via luciferase activity in all constructs based on

pFS7. Luciferase activity was measured using the Luciferase Assay System from Promega, according to the manufacturer’s protocol. The luminometer used was a GloMax 20/20 (Promega). Strains were grown as described above. RNA isolation, cDNA synthesis and qRT-PCR Transcript amounts were determined by two-step quantitative selleck compound real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). RNAqueous (Ambion) was used for total RNA isolation. Isolated RNA was treated with Turbo DNAse (Ambion) and reverse transcription was performed using a first-strand cDNA synthesis kit with random pd(N)6 primers (Amersham Biosciences). PCR was carried out in the presence of Power SYBR Green PCR Master Mix (Applied Biosystems) using a 7500 Real Time PCR system (Applied Biosystems).

During PCR samples were heated to 95°C for 10 min, followed by 40 cycles of amplification (95°C for 15 s; 60°C for 1 min). Results were analysed by 7500 system Tozasertib supplier software v1.3 using the 2-∆∆CT method [39]. Primers were designed using Primer Express software (Applied Biosystems). For xylS primers 5′-TGTTATCATCTGCAAATAATACTCAAAGG-3′ and 5′-GCCCGGCGCAAAATAGT-3′ were used. 16S rRNA was used as endogenous control with the primer pair 5′-ATTGACGTTACCCGCAGAAGAA-3′ and 5′-GCTTGCACCCTCCGTATTACC-3′. Protein analysis by SDS-PAGE For SDS-PAGE analysis cells were grown in a volume of 25 mL. Cultures containing plasmid pET16b.xylS were induced with 0.5 mM IPTG or grown in the absence

of inducer. After centrifugation the pellets were washed in 0.9% NaCl. 100 mg pellet (wet weight) were resuspended in 0.5 mL lysis buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0, 20% sucrose), 1 mg lysozyme and 62.5 U mL-1 benzonase nuclease (Sigma) were added and samples were left with shaking at triclocarban room temperature for 2 hours. After centrifugation (13.000 rpm, 8 min) the supernatant was used as soluble fraction, while the pellet was resuspended in 0.5 mL SDS-PAGE running buffer, giving the insoluble fraction. Protein gels were run under denaturing conditions using ClearPAGE 10% gels and ClearPAGE SDS-R Run buffer (C.B.S. Scientific) followed by staining with Coomassie Brilliant blue R-250 (Merck). References 1. Brautaset T, Lale R, Valla S: Positively regulated bacterial expression systems. Microb Biotechnol 2009, 2:15–30.PubMedCrossRef 2. Mergulhão FJM, Monteiro GA, Cabral JMS, Taipa MA: Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. Microbiol Biotechnol 2004, 14:1–14. 3. Ramos JL, Marques S, Timmis KN: Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators.