Plasmid profiles The plasmid profiles of four transconjugants from each cross were visualized on agarose gels according to the protocol described by Hynes and McGregor [26]. DNA isolation and manipulation Plasmid DNA was isolated using the High Pure Plasmid Isolation Kit (Roche) according to the manufacturer’s
instructions. Restriction and ligation reactions were performed under the conditions PD0332991 in vivo specified by the enzyme manufacturer (Fermentas). PCR was performed using Platinum High Fidelity Taq Platinum Polymerase or ThermalAce™ DNA Polymerase (Invitrogen). PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen). Plasmid incompatibility The incompatibility properties of the constructs were determined as described in Ramírez-Romero et al. [7]. Plasmid replication in R. etli To determine the replication capabilities of the pDOP derivatives in R. etli, the plasmids were introduced into CFNX107 by conjugation. The plasmid profiles of at least four transconjugants from each cross were analyzed. A recombinant plasmid was considered capable of replicating in R. etli if the plasmid profiles of the transconjugants showed a new band of the expected size. Plasmid copy-number determination
The plasmid copy numbers of the CFNX107 transconjugants containing pDOP derivatives were evaluated as follows: the total DNA of each transconjugant was isolated, digested with HindIII endonuclease, resolved in a 1% agarose gel and transferred to Hybond-N+ membranes (Amersham). The blot was then simultaneously hybridized with an Ω- spectinomycin cassette Oxymatrine located within the recA gene (chromosome-encoded) and with a fragment of pDOP; both probes were of the same size and GC MK-4827 cost content. The hybridization signals were CUDC-907 datasheet quantified with a PhosphorImager SI (Molecular Dynamics). The plasmid copy-number was calculated from the ratio of the integrated hybridization signal of the recombinant plasmid and the integrated hybridization signal of the chromosome. Bioinformatics Alignments were performed with Clustal-W [27] at the WWW service of the European Bioinformatics Institute http://www2.ebi.ac.uk/clustalw. Protein secondary structure predictions were made with PSIPRED [28] at the WWW service of
the Bioinformatics Group, UCL Department Of Computer Science http://bioinf.cs.ucl.ac.uk/psipred/. The DNA duplex helical stability profile was calculated using WEB-THERMODYN: sequence analysis software for profiling DNA helical stability http://www.gsa.buffalo.edu/dna/dk/WEBTHERMODYN/[29]. Results The oriV of p42d is located within the repC coding sequence The basic replicon of Rhizobium etli p42d, defined as the smallest DNA region that contains all of the elements required to replicate with the same stability and plasmid copy-number as the parental plasmid, consists of the complete repABC operon plus 500 bp downstream of the repC stop codon (inc-beta region, containing parS) and 86 bp upstream of the repA initiation codon [8] (Figure 1).