characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-05-01T17:02:13Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-04-06T15:14:28Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA103959741
characteristics.characteristic: External Id
characteristics.text: Department of Biochemistry and Molecular Biology, University of Southern Denmark
characteristics.characteristic: INSDC center name
characteristics.text: 2017-05-01T17:02:13Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-04-06T15:14:28Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1648909
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5657:DMEM
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Protocols: Plasmid transformed UTI89/pMWLib culture was grown at 37 °C for 1 hour without aeration in preheated DMEM with 10 % FBS containing 100 μg/mL ampicillin (amp) for plasmid selection. UTI89/pMWLib cells emitting green fluorescence were collected by cell sorting, using an FACSAria II. The bacteria were then collected by centrifugation at 3500xg for 10 minutes, resuspended in LB and grown overnight. The UTI89 promoter trap library was constructed as described by Bumann and Valdivia (Nat Protoc 2007, 2: 770-777) with minor modifications. Chromosomal DNA was purified from an overnight culture of UTI89. Bacterial cells were harvested and resuspended in TE buffer (0.089 % Trizma hydrochloride (w/v), 0.053 % Trizma base (w/v) and 0.1 mM EDTA), rapid lysis buffer (5 % sodium dodecyl sulfate (SDS), 0.125 M EDTA, 0.5 M Tris-HCl pH 9.4), Ribonuclease A (Sigma Aldrich) and protease (Roche) (37°C) and incubated for one hour at 37 °C. Chromosomal DNA was purified by phenol-chloroform extraction, precipitated by addition of 2 vol. 96% ethanol, and resuspended in TE-buffer. Purified genomic DNA (100ng/ml) was disrupted by sonication for 2x30 seconds using a Branson Sonifier 250 (Branson Ultrasonics). Resulting fragments of 500-700 bp were purified from a 2% agarose gel using a GFX PCR DNA and Gel Band purification kit (GE Healtcare) and treated with T4 polymerase (New England Biolabs) for 15 minutes at 12 °C to repair single-stranded regions. The library vector pMW82 was cleaved with BamHI (Fermentas) and SphI (Fermentas), and treated with T4 polymerase to fill in single-stranded overhangs. Next, treatment with shrimp alkaline phosphatase (USB Corporation) was included to prevent vector re-ligation. Finally, genomic fragments and vector DNA was ligated using T4 DNA ligase. Ligated DNA was introduced into MegaX DH10BTM T1R ElectrocompTM Cells (Invitrogen) by electroporation, and transformants were selected by plating on LB agar containing 100 μg/ml amp. The frequency of vector re-ligation was estimated to be less than 20 %. Plasmids were subsequently purified using NucleoBond AX kit (Macherey-Nagel) and transformed into E. coli UTI89 by electroporation. The resulting amp-resistant colonies, together constituting the promoter trap library UTI89/pMWLib, were pooled and frozen at -80°C. Plasmids were purified from sorted UTI89/pMWLib using NucleoBond PC 500 kit (Macherey-Nagel) as specified by the manufacturer. PCR amplification of the inserted chromosome segments in pMWLib was performed using primers P1 (5’-TGAAGGCTCTCAAGGGCATC-3’) and P2 (5’-GTGTTGGCCATGGAACAGGT-3’). DNA fragments were sonicated into 250-400 bp long segments from the original size of 500-700 bp, and sequencing library preparation was performed according to the manufacturer’s instructions (Illumina) as described previously (Nielsen R, Mandrup S. Methods Enzymol 2014, 537: 261-279).
characteristics.characteristic: description
characteristics.text: DMEM medium
characteristics.characteristic: growth condition
characteristics.text: J82 Bladder Epithelial Cells (BECs)
characteristics.characteristic: host
characteristics.text: Escherichia coli UTI89
characteristics.characteristic: organism
characteristics.text: Escherichia coli UTI89
characteristics.characteristic: organism
characteristics.text: Escherichia coli UTI89
characteristics.characteristic: scientific_name
characteristics.text: Plasmid transformed UTI89/pMWLib
characteristics.characteristic: strain
characteristics.text: DMEM
characteristics.characteristic: title