Journal: PLoS ONE
Article Title: Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics
Figure Lengend Snippet: Construction of pKS-VOTL plasmid. (A) Organisation of spo0B operon in B . subtilis and putative obg operon in M . synoviae has been shown. Putative –10 promoter region, transcription start site, ribosome binding site (RBS) of vlhA promoter region, and initiation codon for vlhA gene have been indicated. Length (bp) of each CDS is indicated inside the arrows. Identified stem loop structures in B . subtilis spo0B operon and putative stem loop in M . synoviae ‘ obg operon’ have also been indicated. (B) Schematic presentation of splicing by overlap extension (SOE) PCR to join vlhA gene promoter with obg CDS. Using PCR#1 and PCR#2, vlhA promoter (solid lines) and obg CDS (dotted lines) were amplified using indicated primers. Intermediate products with overlapping fragments (shown by horizontal bars) from both PCRs were amplified by SOE-PCR (PCR#3) using primers vlhA-ExtF and obg-ExtR. (C) Agarose gel electrophoresis of amplification products of vlhA promoter region PCR, obg CDS PCR and SOE-PCR. MW, DNA molecular weight marker (PCR Marker, Sigma, Missouri, USA). (D) Final product of SOE-PCR was first cloned at T-site of pGEM-T Easy Vector and then Pst I- Sac II fragment containing vlhA - obg was inserted between Pst I and Sac II sites of pBluescript II KS (+) vector. Apa I- Sal I restriction fragment containing LoriC and tetM , from pMAS-LoriC plasmid, was cloned at respective sites in pBluescript II KS (+) vector, harboring vlhA - obg , to generate pKS-VOTL plasmid.
Article Snippet: Amplicons of expected size were confirmed by agarose gel electrophoresis ( ) and vlhA - obg amplicon was cloned into pGEM-T Easy vector according to the manufacturer’s instructions (Promega, Alexandria, New South Wales, Australia).
Techniques: Plasmid Preparation, Binding Assay, Overlap Extension Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker, Clone Assay