Corynebacterium glutamicum
Protocols: Strain ATCC 13032, harvesting cells after cultivation and/or sorting using a FACS device. In general, counter-silencing covers mechanism that are used to overcome repressing activities of xenogeneic silencers like H-NS, Lsr2 or here CgpS. One way to achieve counter-silencing of CgpS activity is to use a truncated version of CgpS itself consisting only of the oligomerization domain (as described in Pfeifer et al., 2016, doi: 10.1093/nar/gkw692). Here we used 150 µM IPTG to overexpress the truncated version. For the counter-silencing experiments the expression of an genomically integrated eyfp was monitored by flow cytometry and sorted by FACS. The eyfp gene was cloned under a phage promoter called lys (for lysin), which can be activated by the counter-silencing mechanism (described below). For large volumes: RNeasy Kit (Qiagen, Hilden, Germany), cell disruption was performed with glass beads For small volumes (sorted samples): NucleoZol (Machery-Nagel, Düren, Germany) was used, cell disruption was performed with lysozyme + mutanlysin large sample volumes: rRNA depletion was done using Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) (Epicentre/illumina, Munich, Germany) small volumes: a Protocol for Removal of rRNA from Small Amounts of Total RNA” from Clontech used All libraries were generated using: TruSeq Stranded emRNA Library Prep Kit (Illumina, Munich, Germany)
ENA-FIRST-PUBLIC
2018-09-15T17:01:47Z
ENA-LAST-UPDATE
2018-05-29T15:57:37Z
External Id
SAMEA4697066
INSDC center name
IBG-1, Forschungszentrum Julich GmbH
INSDC first public
2018-09-15T17:01:47Z
INSDC last update
2018-05-29T15:57:37Z
INSDC status
public
Submitter Id
E-MTAB-6809:Sample 12
broker name
ArrayExpress
genotype
IPTG-inducible expression of truncated CgpS; lys-EYFP
phenotype
unsorted
replicate
3
sample name
E-MTAB-6809:Sample 12
scientific_name
Corynebacterium glutamicum
stimulus
counter-silencing
strain
ATCC 13032
type-material
type strain of Corynebacterium glutamicum