Journal: PLoS ONE
Article Title: Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis
Figure Lengend Snippet: Sequence characteristics of transforming DNA. A. Variable DUS positions and restriction profiles in transforming DNA plasmids. Transforming DNA plasmids are based on pilG (black) interrupted by an erythromycin resistance insert, ermC , (gray). DUS ( 5′- ATGCCGTCTGAA-3′ ) or reverse complimentary DUS ( 5′-TTCAGACGGCAT-3′ ) are inserted in three different positions A, B and C as marked above the bar. All numbers refer to the nucleotide position following the start codon (1) of pilG. The 137 nucleotide long Bam HI-fragment which is removed in pDV-b, pDV-c versions is shown in white with black stripes. B. Nla IV restriction profile of rpoB and location of selective SNP. The homologous 723 nt long PCR fragment of an internal part of the meningococcal rpoB gene used in transformation contains two Nla IV restriction sites on both sides of the selective SNP responsible for rifampicin resistance in the recipient.
Article Snippet: The hybrid plasmids pOHA-0-DUS and pOHA-Single from a previous study containing the naturally DUS-less neisserial pilus biogenesis gene pilG harbouring a selective marker encoding erythromycin (erm) resistance , were used to make all constructs employed, varying in DUS, Nla IV sites and homologous region as shown in . pOHA-0-DUS and pSingle were trimmed with Bam HI (recognition sequence GGATCC) (New England Biolabs) and recircularised with T4 Ligase (New England Biolabs) or polished with Phusion polymerase (New England Biolabs) following Bam HI restriction and recircularised to generate the plasmids -b, -c and -d versions of pOHA-0-DUS and pOHA13, respectively.
Techniques: Sequencing, Polymerase Chain Reaction, Transformation Assay