Journal: PLoS ONE

Article Title: Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent

doi: 10.1371/journal.pone.0076341

Figure Lengend Snippet: (A) Schematic representation of the nan genetic organization. Each block arrow indicates an ORF; the length of the arrow is proportional to the length of the predicted ORF. RUMGNA_02702, 02701, 02700, 02699, 02698, 02697, 02695 and 02690 are shown in block arrow to , respectively. Circles above thick vertical lines indicate potential stem-loop structures that might act as Rho-independent transcriptional terminators. The free energy of the thermodynamic ensemble is given on top, expressed as kcal.mol −1 . The inset shows the DNA sequence of the promoter located upstream of the putative RUMGNA_02701 gene ( ). The putative −35 and −10 regions and ribosome-binding site (RBS) are underlined. (B) Confirmation of the nan operonic structure. The PCR products obtained following RT-PCR of RNA extracted from R. gnavus ATCC 29149 grown on pPGM were obtained using primers set spanning the SAT2 to NanK ORFs and analysed by electrophoresis on agarose gel. PCR from RT negative control (RT−) was performed to confirm the absence of genomic DNA contamination of the RNA sample prior to RT. PCR negative (−) and positive (+) controls were carried out with water or ATCC 29149 genomic DNA as template, respectively. The positions of the primers are shown in panel A and their sequences are provided in . M, DNA ladder size marker (with increments indicated in base pairs).

Article Snippet: A total of 1499 60-mer probes were designed for microarray experiments based on R. gnavus ATCC 29149 genome information using Array Designer 3.0 software (PREMIER Biosoft International, Palo Alto, CA) and printed on Agilent Custom Oligonucleotide Microarrays 8×15 k. For sample preparation, the Sau3AI-digested ATCC 29149 genomic DNA (gDNA) and each cDNA were fluorescently labelled using the BioPrime® Array CGH Genomic Labeling System (Life Technologies Ltd) according to supplier's instructions, and Cy3-dUTP or Cy5-dUTP respectively (GE Healthcare UK Ltd, Little Chalfont, UK).

Techniques: Blocking Assay, Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Negative Control, Marker