|
| Status |
Public on Aug 05, 2010 |
| Title |
2.5% vs. 50% air saturation Replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Chemostat culture slow 2.5% air saturation
|
| Organism |
Mycolicibacterium smegmatis MC2 155 |
| Characteristics |
culture doubling time: 69 hours
|
| Growth protocol |
Prior to each experiment bacteria were loop inoculated from a cryo-culture (-80°C) onto a LBT plate and incubated for 3 days at 37°C. A colony was picked from the plate and inoculated into the 5 ml batch medium in a 30 ml glass universal and incubacted at 37°C on a rotary shaker at 200 rpm. At an OD between 0.3 - 0.5 a sample was withdrawn to inoculate the bioreactor (700 ml) to a starting OD600 of 0.005. The culture was then left in batch mode under oxystatic condition (maintaining an air saturation of 50% i.e. 10% dissolved oxygen concentration) until OD600 reached around 80% of maximum OD600 and then turned in to chemostat mode either at a dilution rate of 4.6 h-1 or 69 h-1. The chemostat was then left running for at least two volume changes before harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA were reverse transcribed and labelled in a two step protocol according to SOP M007 from TIGR (Hegde, P., Qi, R., Abernathy, K. & other authors (2000). A concise guide to cDNA microarray analysis. BioTechniques 29, 548-550, 552-544, 556 passim.)
|
| |
|
| Channel 2 |
| Source name |
Chemostat culture slow 50% air saturation
|
| Organism |
Mycolicibacterium smegmatis MC2 155 |
| Characteristics |
culture doubling time: 69 hours
|
| Growth protocol |
Prior to each experiment bacteria were loop inoculated from a cryo-culture (-80°C) onto a LBT plate and incubated for 3 days at 37°C. A colony was picked from the plate and inoculated into the 5 ml batch medium in a 30 ml glass universal and incubacted at 37°C on a rotary shaker at 200 rpm. At an OD between 0.3 - 0.5 a sample was withdrawn to inoculate the bioreactor (700 ml) to a starting OD600 of 0.005. The culture was then left in batch mode under oxystatic condition (maintaining an air saturation of 50% i.e. 10% dissolved oxygen concentration) until OD600 reached around 80% of maximum OD600 and then turned in to chemostat mode either at a dilution rate of 4.6 h-1 or 69 h-1. The chemostat was then left running for at least two volume changes before harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA were reverse transcribed and labelled in a two step protocol according to SOP M007 from TIGR (Hegde, P., Qi, R., Abernathy, K. & other authors (2000). A concise guide to cDNA microarray analysis. BioTechniques 29, 548-550, 552-544, 556 passim.)
|
| |
|
| |
| Hybridization protocol |
Hybrization performed according to SOP M008 from TIGR (Hegde, P., Qi, R., Abernathy, K. & other authors (2000). A concise guide to cDNA microarray analysis. BioTechniques 29, 548-550, 552-544, 556 passim.)
|
| Scan protocol |
Scanned Genepix 4000A scanner .
Images were quantified using Spotfinder (TIGR).
|
| Description |
Biological replicate 1 of 5.
|
| Data processing |
Total normalization and LOWESS normalization with MIDAS software (TIGR)
|
| |
|
| Submission date |
Aug 05, 2009 |
| Last update date |
Aug 05, 2009 |
| Contact name |
Michael Berney |
| Organization name |
Albert Einstein College of Medicine
|
| Department |
Department of Microbiology and Immunology
|
| Lab |
Jacobs lab
|
| Street address |
1301 Morris Park Avenue
|
| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
| |
|
| Platform ID |
GPL5862 |
| Series (1) |
| GSE17520 |
M. smegmatis response to energy- and oxygen-limitation in continuous culture |
|
