Project description:The DNA content of bacteriophages from Bartonella grahamii was investigated by hybridization against cellular DNA from the same organism. Phage particles were isolated from plate grown bacteria as well as from different growth phases during culture in liquid medium.
Project description:Bacteriophages are potent therapeutics against biohazardous bacteria that are rapidly acquiring multidrug resistance. However, routine administration of bacteriophage therapy is currently impeded by a lack of safe phage production methodologies and insufficient phage characterization. We thus developed a versatile cell-free platform for host-independent production of phages targeting gram-positive and gram-negative bacteria. A few microliters of a one-pot reaction produces effective doses of phages against potentially antibiotic-resistant bacteria such as enterohemorrhagic E. coli (EAEC) and Yersinia pestis, which also possibly pose threats as biological warfare agents. We also introduce a method for transient, non-genomic phage engineering to safely confer additional functions, such as a purification tag or bioluminescence for host detection, for only one replication cycle. Using high-resolution and time-resolved mass spectrometry, we validated the expression of 40 hypothetical proteins from two different phages (T7 and CLB-P3) and identified genes in the genome of phage T7 that express exceptionally late during phage replication. Our comprehensive methodology thus allows for accelerated reverse and forward phage engineering as well as for safe and customized production of clinical-grade therapeutic bacteriophages.
Project description:We used single-cell whole genome sequencing (scWGS) to assess aneuploidy in isolated neurons from the frontal cortex of control individuals. This experiment is related to E-MTAB-4184, which contains Alzheimer's disease samples.
Project description:This study analysed the temporal transcriptional response of L. lactis UC509.9 undergoing infection with either Tuc2009 or c2, representing phages of two different species (P335 and c2, respectively) of the family Siphoviridae. For the first time, to our knowledge, both DNA microarrays of the host and high resolution tiling arrays of each phage were used provide corresponding data sets of the entire transcriptome at various points post-infection. DNA microarrays containing oligonucleotide primers representing each of the 2066 annotated genes on the genome of L. lactis UC509.9 (Genbank accession number: CP003157), in addition to complete genome tiling arrays of bacteriophages Tuc2009 NC_002703) and C2 (NC_001706) at 4 bp resolution, were designed using eArray (https://earray.chem.agilent.com/earray/) and ChipD (http://www.ncbi.nlm.nih.gov/pubmed/20529880) and obtained from Agilent Technologies (Palo Alto, CA). For sample collection, pre-warmed GM17 (30 M-BM-0C) was inoculated with 2 % of an overnight culture of L. lactis UC509.9. This was grown at 30 M-BM-0C under static conditions to an OD600 of 0.13 at which point CaCl2 was added to a final concentration of 10 mM. The culture was further incubated for 10 min to allow equilibration. At this point, the culture was split into two equal volumes. To one, phage (either C2 or Tuc2009) in TBT buffer (100 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2), was added to a final multiplicity of infection (MOI) of 5. To the other, acting as control, a corresponding amount of TBT buffer without phage was added. Samples (60 ml) were collected at 2, 5, 10, 15, 25, 35 and, in the case of Tuc2009 only, 45 min post infection (p.i.) by centrifugation. Pellets were flash frozen in a -80 M-BM-0C EtOH bath. Samples were then maintained at -80 M-BM-0C until further processing and analysis.
Project description:We used single-cell whole genome sequencing (scWGS) to assess aneuploidy in isolated neurons from the frontal cortex individuals with mild AD (Braak stage III) and individuals with advanced AD (Braak stage VI). This experiment is related to E-MTAB-4185, which contains control samples.
