The supernatant obtained selleck chemicals llc after centrifugation (14,000 x g, 10 min) was used directly as template for quantitative (real time) PCR analyses. Quantitative real time PCR analysis Plasmid copy numbers were determined by quantitative real time PCR (qPCR) using a relative quantification approach, based on the procedure

described by Skulj et al.[42]. qPCR was performed in 20 μl reaction mixtures in MicroAmp optical 48-well reaction plates, using the Fast SYBR Green PCR Master Mix reagent (Applied Biosystems, CA, USA) on a StepOnePlus Real-Time PCR system (Applied Biosystems, CA, USA) controlled by StepOne Software Version 2.0 (Applied Biosystems). Primers were designed using Primer Express Software Version 3.0 (Applied Temozolomide chemical structure Biosystems; see Additional file 1 for qPCR primer sequences). Plasmid DNA concentrations were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, DE, USA). Serial dilutions of the pUCZM-1 and pUCZM-3 plasmids were used to create Vadimezan chemical structure standard curves for quantifying pZMO1A and pZMO7 plasmid concentrations. A pCR2.1 TOPO vector containing the PCR-amplified polyphosphate kinase 2 (ppk2, ZZ6_0566) gene from Z. mobilis ATCC 29191 (ppk2-TOPO) was similarly used to construct a standard curve for Z. mobilis chromosome copy number determination. Concentrations of chromosome molecules, native plasmids and recombinant

plasmids were individually quantified by qPCR within aliquots from the same freshly-prepared cell lysate supernatants prepared from wild-type or transformed Z. mobilis strain cultures (as described

above). The (relative) plasmid copy numbers (PCNs) in each sample were calculated by dividing the concentration of the respective plasmid molecules by the concentration of chromosome molecules. All qPCR experiments were performed in duplicate, with at least two independent biological replicates. Analysis of pZ7C plasmid-based Glutathione S-Transferase (GST) and GST fusion protein expression in E. coli and Z. mobilis Freshly-transformed starter cultures of recombinant E. coli BL21 (DE3) strains containing the pZ7-GST, pZ7-GST-acpP, pZ7-GST-dnaJ, pZ7-GST-hfq, pZ7-GST-holC or pZ7-GST-kdsA plasmids PJ34 HCl in LB media containing 30 μg/ml Cm were expanded 1:50 into fresh LB containing 30 μg/ml Cm (800 ml) and grown aerobically with shaking (37°C) until OD600nm of ca. 1.0. Cultures were chilled in ice-water, and cell pellets were collected by centrifugation (4,000 x g, 10 mins 2-4°C), washed with 10% aqueous glycerol, then resuspended in 20 ml ice-cold binding buffer (25 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 1.5 mM beta-mercaptoethanol). Cells were lysed by sonication with ice-cooling (Sonics Vibra-Cell, 40% amplitude; 5 cycles of: 3 s pulse-on, 9 s pulse-off; 1 min). After centrifugation (12000 x g, 30 mins, 4°C), the supernatant was filtered (0.45 μm syringe filter, Iwaki Co., Ltd.