Dataset features


Application: Gene expression microarray analysis
Number of samples: 36
Release date: Jul 31 2007
Last update date: Jan 15 2017
Access: Public
Chemicals: Oxygen
Dataset link Halobacterium NRC-1_oxygen_response_timecourse_set3

Experimental Protocol

This experiment was a replicate of Oxygen time series 1, except that cells were equilibrated at low oxygen for 24 hours prior to the experiment start. Also, time course sampling was more frequent after the shift to high oxygen and sampling was continued beyond 6 hours to 12 hours. Halobacterium sp. NRC-1 (ATCC700922) was routinely grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light. For turbidostat experiments, starter cultures of NRC-1 were inoculated into 2L of CM in a 3.0 L vessel (5-10% inoculum) and grown to mid-logarithmic phase (OD600 ~ 0.5) in batch mode in a BioFlo100 modular bench top fermentor (New Brunswick Scientific, Edison, NJ) at 300 rpm, pH 7.0. Prior to each experiment, an oxygen sensor (model InPro 6000, Mettler Toledo, Columbus, OH) was calibrated to 100% oxygen at 1200rpm and sparging with 3.2 VVM of air. These conditions were approximately equivalent to oxygen saturation in CM medium, which is 1.6 mg/L (~5uM). Once the culture reached mid-logarithmic phase, the oxygen level was rapidly decreased to ~0-0.5% within 5-10 minutes (achieved by cutting off airflow and decreasing agitation to 250 rpm), and allowed to incubate under anoxic conditions for 24 h prior to the start of sampling. The oxygen concentration in the culture was then rapidly increased to approximately 85-100% (achieved by agitating at 1100 rpm and sparging with air at 3.2 VVM) and sampling commenced and continued at the times indicated in the Sample records. The culture was subsequently maintained in high oxygen conditions for 12hr. During these perturbations, all other parameters were kept constant (pH 7.2-7.3, 37ºC, ambient light, O.D.600~0.5-6.5). Each culture sample taken at the times indicated in the Sample records was split in half, one half being used for RNA extraction and the other for ATP measurement.








Amy Schmid