|
| Status |
Public on Feb 14, 2015 |
| Title |
0186_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
0186 under carbon starvation rep1
|
| Organism |
Gordonia sp. KTR9 |
| Characteristics |
genotype/variation: KTR9_0186 disruptant
exposed to: carbon starvation
|
| Growth protocol |
Gordonia sp. KTR9 and KTR9 mutant 0186 were grown in mineral salts media supplemented with succinate (10 mM), gluconate (10 mM), fructose (10 mM) and ammonium chloride (18 mM) for 72 h at 30°C shaking at 150 rpm. This was used as a starter culture to inoculate triplicates of KTR9 and KTR9 mutant 0186.
Triplicates of KTR9 and KTR9 mutant 0186 were inoculated at an OD600 of 0.015 and grown in mineral salts media supplemented with succinate (10 mM), gluconate (10 mM), fructose (10 mM) and ammonium chloride (18 mM) for 72 h at 30°C shaking at 150 rpm.
For nitrogen starvation, cultures were spun down, rinsed in PBS buffer, and resuspended in mineral salts media with succinate (10 mM), gluconate (10 mM), and fructose (10 mM) and incubated at 30C for 48h.
After nitrogen starvation, cultures were exposed to carbon starvation. Cultures were spun down, rinsed in PBS buffer, and resuspended in mineral salts media with ammonium chloride (18 mM) and incubated at 30C for 72h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and according to the manufacturer’s instructions for RNA cleanup with an added enzymatic lysis step and mechanical disruption step added before cleanup. Enzymatic lysis and mechanical disruption was performed by resuspending the cell pellet in 100 uL TE buffer and 200 uL lysozyme (20 mg/mL), transferring the resuspended pellet to a MP Biomedicals (Solon, OH) Lysing Matrix B tube and bead beating for 30s. The optional on column DNA digestion using the Qiagen RNase-Free DNase Kit was performed to remove DNA. RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer. The RNA was used to generate cDNA using the Superscript II Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s directions. At each step the quality of the RNA and cDNA was analyzed using the Agilent 2100 Bioanalyzer and DNA 1000 Kit (Agilent).
|
| Label |
Cy3
|
| Label protocol |
The cDNA was labeled with the One-Color DNA Labeling Kit (Nimblegen, Madison, WI) and the concentration verified using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA).
|
| |
|
| Hybridization protocol |
Two micrograms of the labeled DNA was used for subsequent hybridization to custom Nimblegen Bacterial Gene Expression Arrays for Gordonia sp. KTR9. The hybridized arrays were washed using the Nimblegen Wash Buffer Kit.
|
| Scan protocol |
Washed arrays were scanned using the Agilent Microarray Scanner
|
| Description |
0186 A
This sample is of Gordonia sp. KTR9 0186 Mutant under carbon starvation conditions. It is the first of three biological replicates used in this experiment, each from separate cultures.
|
| Data processing |
Microarray images were analyzed using Nimblescan v2.5.26 software (Nimblegen) and ArrayStar v4.0.0 (DNAstar, Madison, WI) software. Expression data were log2 transformed and statistical significance was determined using a moderated t-test for binary transcriptome comparisons (i.e. control vs. experimental). The P-value was set at 0.05 and only those genes induced or repressed at least fivefold were considered in this paper. Differentially expressed transcripts were assigned to their functional classifications based on the Comprehensive Microbial Resource genome annotation from the J. Craig Venter Institute.
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| |
|
| Submission date |
Aug 08, 2014 |
| Last update date |
Feb 14, 2015 |
| Contact name |
Dawn E Hancock |
| E-mail(s) |
dawn.hancock@usace.army.mil
|
| Phone |
6016343711
|
| Organization name |
DEPARTMENT OF DEFENSE Army Corps of Engineers
|
| Department |
Engineer Research and Development Center
|
| Lab |
Environmental Lab
|
| Street address |
3909 Halls Ferry Road
|
| City |
Vicksburg |
| State/province |
Mississippi |
| ZIP/Postal code |
39180 |
| Country |
USA |
| |
|
| Platform ID |
GPL16292 |
| Series (1) |
| GSE60245 |
The effects of putative lipase and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase gene knockouts on triacylglycerol accumulation and transcriptome expression in Gordonia sp. KTR9. |
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