BioProject

A L. acidophilus NCFM mutant (NCK1909) carrying an in-frame deletion within the upp gene (LBA0770) was constructed as a background host for a newly developed upp-based counterselectable gene replacement system for L. More...

A L. acidophilus NCFM mutant (NCK1909) carrying an in-frame deletion within the upp gene (LBA0770) was constructed as a background host for a newly developed upp-based counterselectable gene replacement system for L. acidophilus. Since the NCK1909 mutant serves both as the host for genetic exchange and the reference strain for subsequent phenotypic studies of deletion mutants that will be generated from the counterselective gene replacement system, comparative gene expression study was performed to ensure the genotype of NCK1909 is representative of the NCFM parent strain. Overall design: Oligoarray hybridization experiments were performed to compare the transcriptional profiles of the NCK1909 mutant with relative to NCFM at early-log (OD600nm 0.3) and mid-log (OD600nm 0.8) phases when cultured in MRS medium at 37 C under ambient atmospheric condition. These cell density levels represent the growth stages when cells were collected for stress challenge assays (OD600 0.25 to 0.3) and in vitro cell adherence assays (OD600 0.6 to 0.8) during phenotypic analyses. NCFM and NCK1909 were grown in fresh MRS medium with a 2% inoculum from overnight culture. Aliquots of cells were collected at OD600nm of 0.3 (early log-phase) and 0.8 (mid-log phase) for total RNA extraction and cDNA synthesis. Samples were collected from two independent experimental replicates (representing two biological replicates, designated as Rep A and Rep B). Labeled cDNA samples from NCFM and NCK1909 were coupled with mono-reactive Cy3 and Cy5 dyes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), respectively. Comparative hybridizations were performed on samples representing both NCFM and NCK1909 at same growth phases. Less...