Derived from Actinosynnema pretiosum (ATCC:31565) Resistant to 50ug/mL rifampicin
Growth protocol
Spores from single colonies were cultured in Vegetative medium (5 g/l meat extract, 5/l g peptone, 5 g/l yeast extract, 2.5 g/l enzyme hydrolysate of casein, 20 g/l glucose, 1.5 g/l NaCl) for 2 days at 26oC. 2mL of seed culture was used to innoculate 320mL of YMG media (4 g/l yeast extract, 10 g/l malt extract, 4 g/l glucose, pH 7.2) Cultures were incubated at 26oC shekn at 190rpm. Samples were taken at day 2, 4, 6 and 8 of the culture. The samples were centrifuged at 3000g at 4oC for 15 mintues. The supernatent was used for Ansamitocin-P3 analysis and the volume of the cell pellet estimated(PCV) before freezing and storing at -80oC.
Extracted molecule
total RNA
Extraction protocol
DEPC treated water equvilent to 0.5 x volume of cell pellet was added to the cell pellet. After cell pellet has thawed, it is subjected to 5 more cycles of freezing thawing in liquid nitrogen and a 55oC waterbath respectively. The cell pellet is then treated with lysozyme (3.5 mg/ml) at 37oC for 20 min. The supernatent removed through centrifugation and the pellet mixed with Rnawiz (Ambion, USA) and RiboPureTM-Bacteria Kit (Ambion, USA) was used as per manufecturer's protocol. Optional Dnase cleanup step was also performed.
Label
Cy3
Label protocol
10ug of total RNA was incubated with 2ug of randomer hexamer. Incubated at 70Co for 5 mintues and placed on ice. 1pmole of Cy3 labelled dUTP, low dT-dNTP mix Rnase inhibitor and 2uL of RevertAidTM H- Minus M- MuLV Reverse Transcriptase (Fermentas Life Sciences, MD) was added. Mixture is incubated at room temperature for 10 minutes folloed by 42Co for 2 hours 10 µg of total RNA were primed with 2 µl of 100 µM random DNA primer (N9) at 70°C for 5min, then reversed transcribed at 42°C for 1 h in the presence of 400 U RevertAidTM H- Minus M- MuLV Reverse Transcriptase (Fermentas Life Sciences, MD), and 25 µM each dATP, dCTP, dGTP, with 12.5 µM dTTP, and 25 µM Cy3-labelled dUTP.
Spores from single colonies were cultured in Vegetative medium (5 g/l meat extract, 5/l g peptone, 5 g/l yeast extract, 2.5 g/l enzyme hydrolysate of casein, 20 g/l glucose, 1.5 g/l NaCl) for 2 days at 26oC. 2mL of seed culture was used to innoculate 320mL of YMG media (4 g/l yeast extract, 10 g/l malt extract, 4 g/l glucose, pH 7.2) Cultures were incubated at 26oC shekn at 190rpm. Samples were taken at day 2, 4, 6 and 8 of the culture. The samples were centrifuged at 3000g at 4oC for 15 mintues. The supernatent was used for Ansamitocin-P3 analysis and the volume of the cell pellet estimated(PCV) before freezing and storing at -80oC.
Extracted molecule
genomic DNA
Extraction protocol
DEPC treated water equvilent to 0.5 x volume of cell pellet was added to the cell pellet. After cell pellet has thawed, it is subjected to 5 more cycles of freezing thawing in liquid nitrogen and a 55oC waterbath respectively. The cell pellet is then treated with lysozyme (3.5 mg/ml) at 37oC for 20 min. The supernatent removed through centrifugation and the pellet mixed with Rnawiz (Ambion, USA) and RiboPureTM-Bacteria Kit (Ambion, USA) was used as per manufecturer's protocol. Optional Dnase cleanup step was also performed.
Label
Cy5
Label protocol
Actinosynnema pretiosum genomic DNA was sheared by sonicating on ice with the Vibra-CellTM fitted with a 3mm probe (Sonics and Materials, CT) for 1 min. 0.1ug of sheared gDNA was incubated with 300µg/mL random DNA octomers at 96Co for 5 mintues then placed on ice. It was then incubated with 50U of klenow fragments (New England Biolabs, USA), 120µM each of dATP, dCTP,dGTP, 60µM of dTTP and 40µM of Cy5-labelled dUTP at 37oC for 2 hours.
Hybridization protocol
Slides were pre-hybridized with 5x saline sodium citrate buffer(SSC), 50% deionised formamide, 0.1% sodium dodecyl sulphate(SDS) and 1 % Bovine serum albumin for 2 hour at 42oC. Labelled cDNA and gDNA was cleaned up using mineluate columns per manufacture’s protocol (Qiagen, USA). Hybridisation mixture comprising of labelled cDNA from 10ug of total RNA, 0.1ug of labelled gDNA, 5 x SSC, 25% deionised formamide and 0.1% SDS were heated at 95oC for 5 minutes, cooled on ice before sample was applied to the microarray slides and enclosed in hybridisation chambers for 16 hours at 42oC. Cover slips of the microarray were firsted removed in SSC and 0.2% SDS before being washed in twice in 2 x SSC for 5 mintues each wash, two five minutes wash in 0.1 x SSC with 0.1%SDS and final two washes of 5 mintues each in 0.1x SSC. Slides were subsequently dried by centrifuging at 129g for 3 minutes.
Scan protocol
Slides were scanned on a Axon GenePixÔ 4000B scanner and images were analysed using GenePix Pro 4.1. Expression data was exported into microsoft Excel for normalisation and further analysis
Description
Biological replicate : r50D1 rifampicin resistant clone derived from Actinosynnema pretiocum (ATCC:31565) day 4 sample vs gDNA reference 2 technical repeats
Data processing
Zero or negative value background corrected readings were assigned a value of one. Quantile normalization was carried out based on the method described in Mehra et al. {Mehra, 2006 157 /id}. Briefly, log2(cDNA/gDNA) was first calculated for each data point, before the log ratio of each array was sorted in ascending order and ranked accordingly. The “reference” array was generated by taking the median value of each rank. To normalize the data, percentile values from each of the array to be normalized was replaced by the corresponding value from the reference array before unsorting back to the original order. Technical replicates were averaged and standard deviation was calculated for log intensity ratios for each probe. Probe with log intensity ratios more then 1 standard deviation from the average were treated as outliers and discarded. Technical replicates were then averaged again. Each chip was then scale normalized {Quackenbush, 2002 301 /id} by subtracting the 16s log ratio from each data point.
