Project description:This SuperSeries is composed of the following subset Series: GSE16954: Time course of the mRNA expression after bacteriophage ?YS40 infection in wild-type Thermus thermophilus HB8 strain. GSE16955: Time course of the mRNA expression after phage ?YS40 infection in crp deletion mutant of Thermus thermophilus HB8. Refer to individual Series
Project description:We observed the expression profile of the total mRNA in crp (TTHA1437) deletion mutant strain of Thermus thermophilus HB8 during infection of bacteriophage ÏYS40. Three crp (TTHA1437) delection mutant strain of T. thermophilus HB8 were pre-cultured at 70 C for 16 h in 3 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70°C until A600 value being ~0.8 (1.7 108 cells/ml) that corresponds to logarithmic growth phase. Then the FYS40 phage was infected to the medium at multiplicity of infection (m.o.i.) being ~1, and continued cultivation. Cells were collected after 0, 75 and 100 min infection, and then crude RNA was extracted. The expression of each mRNA at each time point was analyzed on a GeneChip..
Project description:We observed the expression profile of the total mRNA in crp (TTHA1437) deletion mutant strain of Thermus thermophilus HB8 during infection of bacteriophage ϕYS40. Keywords: time course, bacteriophage, infection, CRP, cAMP receptor protein, deletion mutant Three crp (TTHA1437) delection mutant strain of T. thermophilus HB8 were pre-cultured at 70 C for 16 h in 3 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70°C until A600 value being ~0.8 (1.7 108 cells/ml) that corresponds to logarithmic growth phase. Then the FYS40 phage was infected to the medium at multiplicity of infection (m.o.i.) being ~1, and continued cultivation. Cells were collected after 0, 75 and 100 min infection, and then crude RNA was extracted. The expression of each mRNA at each time point was analyzed on a GeneChip as described under Sample Description Sheet of each sample.
Project description:Whole-genome sequencing is an important way to understand the genetic information, gene function, biological characteristics, and living mechanisms of organisms. There is no difficulty to have mega-level genomes sequenced at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. The shotgun sequencing method failed to dissect this genome. After insisting for 10 years and going over 3 generations of sequencing techniques, we successfully dissected the PaP1 genome with 91,715 bp in length. Single-molecule sequencing revealed that this genome contains lots of modified bases, including 51 N6-methyladenines (m6A) and 152 N4-methylcytosines (m4C). At the same time, further investigations revealed a novel immune mechanism of bacteria, by which the host bacteria can recognize and repel the modified bases containing inserts in large scale, and this led to the failure of the shotgun method in PaP1 genome sequencing. Strategy of resolving this problem is use of non-library dependent sequencing techniques or use of the nfi- mutant of E. coli DH5M-NM-1 as the host bacteria to construct the shotgun library. In conclusion, we unlock the mystery of phage PaP1 genome hard to be sequenced, and discover a new mechanism of bacterial immunity in present study. Methylation profiling of Pseudomonas aeruginosa phage PaP1 using kinetic data generated by single-molecule, real-time (SMRT) sequencing on the PacBio RS.