RNA was isolated from three independent cultures of strain B13 grown with 3-chlorobenzoate at exponential phase, early-stationary phase, as well as at 12, 24, 36, 48 and 72 h after the beginning

of stationary phase. Furthermore, duplicate cultures of B13 grown with glucose, fructose and succinate harvested after 24 h, and duplicate cultures grown on succinate in exponential phase were used for RNA purification as well. 15 μl Aliquots of dilutions containing 1, 0.3, and 0.1 μg denatured total RNA were dot-blotted using a 96-well manifold (Gibco Life Technologies) onto positively charged nylon transfer membranes (Hybond-N+, Amersham Biosciences AG). Different concentrations of denatured PCR products (2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01 ng) comprising www.selleckchem.com/products/dibutyryl-camp-bucladesine.html the respective targeted ORF were Selleckchem Obeticholic included on the same blot. RNA was fixed to the membrane with a UV crosslinker before hybridization as described above. Films were scanned and spot intensities were calculated by densitometry using the Image Quant TL program (v2005, Molecular Dynamics, Sunnyville, USA) as grey intensity per standardized surface. The signal intensity of each spot was then compared to the standard curve of Daporinad cell line DNA dilutions on the same blot to calculate an ‘equivalent number of DNA copies’, and divided by the total amount of RNA in the spot to normalize to a value of ‘equivalent

number of copies per μg RNA’. Microarray design A series of 950 non-overlapping 50-mer probes was designed to cover both coding and non-coding regions of the ICEclc sequence (Acc. No. AJ617740) at approximate distances of 200 bp. Probes were designed using the program Oligoarray version 2.1 [36] with a melting temperature range of 92 to 99°C and a probe GC content range of 52 to 72%. Probes were further designed to not cross-hybridize with gene products from the following potential host strains of the ICEclc element: Burkholderia xenovorans LB400 (Acc. No. CP000270-CP000272), P. putida F1 (Acc. No. CP000712), P. putida KT2440 (Acc. No. AE015451),

P. aeruginosa PAO1 (Acc. No. AE004091), Cupriavidus necator JMP134 (Acc. old No. CP000090-CP000093), and Ralstonia metallidurans CH34 (Acc. No. CP000352-CP000355). An additional 93 probes were designed to target housekeeping genes from the potential host strains and 8 probes were designed to target positive/negative controls (GFP, luciferase, and mCherry [37] transcripts). The microarray was manufactured by Agilent Technologies (Santa Clara, CA) in the 8 × 15,000 probe format and each unique probe was synthesized at six randomized spatial locations on the array. The microarray design has been deposited in the NCBI Gene Expression Omnibus http://​www.​ncbi.​nlm.​nih.​gov/​geo under accession number GSE20461. Microarray hybridization and analysis Total RNA was isolated and purified from P.