The suicide plasmid has selleck chemical the R6K origin of replication and encodes resistance to kanamycin and ampicillin. HB101
(pRK600) was used as a helper in triparental mating experiments, providing both resistance to chloramphenicol and the tra function for pUTKm1 mobilization . PCR2.1-TOPO vector was used to clone polymerase chain reaction (pcr) amplification products and transformations performed with One shot® Top10F’ competent E. coli cells, (Invitrogen, California). E. coli strains were grown on Luria Burtani medium at 37°C. Host/plasmid associations were maintained during growth via the incorporation of appropriate antibiotics to media at the following concentrations; 100 μg/ml ampicillin, 25 μg/ml chloramphenicol, 50 μg/ml kanamycin and 20 μg/ml gentamycin. Nucleic acid manipulations Genomic DNA isolation was performed according to Ausubel et al. . Plasmid DNA was isolated from E. coli using a plasmid Miniprep Kit (Qiagen), as per manufacturer’s instructions. DNA visualisations were performed via 1% agarose gel electrophoresis this website in standard TE buffer followed by EtBr staining and photographic capture in a GeneWizard UV trans-illuminator/gel documentation system, (Syngene Bio Imaging). Oligonucleotide primers used in this study were synthesized by Sigma-Genosys, Ltd. (United Kingdom), and are listed
in Table 2. Nucleic acid sequencing was performed by GATC Biotech AG, (Germany), using ABI 3730 × l technology. Routine polymerase Nintedanib (BIBF 1120) chain reactions were carried out in a PTC-200 thermal cycler (MJ Research) using Taq DNA polymerase (Fermentas). High-fidelity amplification requirements were performed with proof-reading, VentR® DNA polymerase (NEB). Table 2 Primers for PCR amplifications. Primer Sequence 5′-3′ Annealing temp°C GS326 acgatgcccagggagtagaga 60 OP2-55 gctgatggcgatgaatgaaca 55 TNInt2 cctgcaggcatgcaagcttcggc 65 27F agagtttgatcatggctcag 55 1492R ggttaccttgttacgactt 55 paaFf paaFr paaGf ggttgagcatgtaggacggt gccaataccgccttgcttga ccgaaggcaactgggtcac 57
57 55 paaGr aggcggcgttcttgttctg 55 paaLf cggcatgctcgcgaccacctg 60 paaLr aaagcgatgttctgcgactc 60 Sig54f-Hind tattacaagcttatgaaaccatcgctgtcctaaaaatga 60 Sig54r-Xba atcatttctagactacatcagtcgcttgcgttcgctcgab 60 paaLproF gccgcgcaacagccagagc 63 paaLproR cgccgagatgccgaggaagg 63 paaLf-Hind tattacaagcttatgacagccctgcgctccttcacctta 60 paaLr-Xba atcatttctagactagtggttactggccttggctb 60 a: Hind III restriction site, b: Xba I restriction site. Oligonucleotide sequences and annealing temperatures utilised in polymerase chain reaction amplification of gene Selleck MAPK inhibitor targets from P. putida CA-3 in this study. Enzyme assays Styrene monooxygenase activity was assessed colorimetrically using whole cell transformations of indole to indigo as previously described . PACoA ligase activity was measured via the method of Martinez-Blanco et al . Activities are expressed as nmol product formed min-1 (mg cell dry weight)-1 for both assays. Cells were harvested at mid-exponential phase unless otherwise stated.