|
Status |
Public on Oct 30, 2009 |
Title |
Evolved Engineered Methylobacterium methanol batch exponential |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
CM1145
|
Organism |
Methylorubrum extorquens AM1 |
Characteristics |
genotype: strain CM1145
|
Biomaterial provider |
Christopher J. Marx
|
Treatment protocol |
10 uL frozen culture were inoculated in 9.6 mL Hypho media supplemented with 15 mM of methanol. The culture was incubated at 30 °C with 225 rpm shaking for 4 days. 0.15 mL culture was then transferred to 9.45 mL of fresh Hypho media and grown under the same conditions for 4 days. From this acclimated culture, 6.25 mL was inoculated into 393.75 mL fresh Hypho media in a 2 liter flask and grown under the same conditions. Bacterial cells were harvested at mid log phase (OD600~0.21).
|
Growth protocol |
10 uL frozen culture were inoculated in 9.6 mL Hypho media supplemented with 15 mM of methanol. The culture was incubated at 30 °C with 225 rpm shaking for 4 days. 0.15 mL culture was then transferred to 9.45 mL of fresh Hypho media and grown under the same conditions for 4 days. From this acclimated culture, 6.25 mL was inoculated into 393.75 mL fresh Hypho media in a 2 liter flask and grown under the same conditions. Bacterial cells were harvested at mid log phase (OD600~0.21).
|
Extracted molecule |
total RNA |
Extraction protocol |
To obtain individual replicates for RNA extraction, samples from cultures were harvested under exponential growth phase and taken as rapidly as possible to limit any potential changes in transcript profiles. A culture volume of 50 mL was sampled directly from the 400 mL batch culture into a conical tube containing 1/10th the culture volume of ice-cold “Stop Solution” (5% water saturated phenol (pH 7.0), 95% absolute ethanol). Cultures were immediately harvested by centrifugation at 4500 g for 8 min at 4°C. Total RNA was extracted from M. extorquens AM1 pellets using the RNeasy Mini Kit (QIAGEN) according to the manufacture’s Yeast III protocol. Lysis was achieved by 3 min of bead-beating in the Mini-Beadbeater 8 (BioSpec Products, Bartlesville, OK) with 600 uL of 0.1-mm zirconium-silica beads (BioSpec Products). SUPERase•In (Ambion, Austin, TX) was added at a final concentration of 1 U/uL to prevent RNA degradation after the elution of RNA with RNase-free water. RNA concentration and purity were determined by using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). RNA integrity was assessed by using a Bioanalyzer (Agilent Technologies) as per manufacturer’s instructions. Residual DNA was removed from isolated RNA samples by treating 60 ug RNA with 36 units of RNasefree DNaseI (Ambion) in a 300-uL reaction volume for 1 h at 37°C. DNaseI-treated total RNA samples were purified by using the RNeasy Mini Kit (QIAGEN) according to the manufacture’s RNA cleanup protocol with a slight modification. Briefly, 1050 uL Buffer RLT and 750 uL ethanol were added to the 300 uL RNA samples and subsequently loaded onto an RNeasy mini column and centrifuged. The remaining RNA sample was successively loaded onto the same column and processed until the rest of the RNA sample was collected on the column. The column was washed three times with 700 uL buffer RW1, and then three times with 500 uL RPE buffer. DNaseI-treated total RNA was eluted with 30 uL of RNase-free water. RNA concentration, purity, and integrity were determined as above. The presence of DNA contamination was examined by PCR. DNaseI-treated total RNA (100 ng) was used as a template in a 50-uL reaction. PCR was done using Taq DNA polymerase (Promega, Madison, WI) with 40 cycles. Only RNA that did not result in a PCR product was used for cDNA synthesis and labeling.
|
Label |
Alexa Fluor 555
|
Label protocol |
The cDNA synthesis and labeling reactions were carried out by using an Amino Allyl cDNA Labeling Kit (Ambion) and Alexa Fluor 555 and Alexa Fluor 647 Reactive Dyes (Invitrogen Corporation, Carlsbad, CA) according to the manufacture’s manual with the following modifications: 5 ug DNaseI-treated total RNA and 4 ug random decamers were used for cDNA synthesis. Ethanol precipitated cDNA incorporating amino allyl dUTP was incubated overnight at 80°C and microcentrifuged for 45 min at 20,800 g at 4°C. Precipitated cDNA was coupled to either Alexa Fluor 555 or Alexa Fluor 647 reactive dye dissolved in 3 uL high-quality DMSO. Dye-labeled cDNA was purified using a QIAquick PCR Purification Kit (QIAGEN) following the instructions in the kit except for performing three washes instead of one. cDNA samples were eluted into 50 uL Nuclease-free water. The incorporation of dyes, the nucleic acid concentration, and the labeling efficiency of dye-labeled cDNA were measured and calculated as described above.
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|
|
Channel 2 |
Source name |
CM702
|
Organism |
Methylorubrum extorquens AM1 |
Characteristics |
genotype: strain CM702
|
Biomaterial provider |
Christopher J. Marx
|
Treatment protocol |
10 uL frozen culture were inoculated in 9.6 mL Hypho media supplemented with 15 mM of methanol. The culture was incubated at 30 °C with 225 rpm shaking for 4 days. 0.15 mL culture was then transferred to 9.45 mL of fresh Hypho media and grown under the same conditions for 4 days. From this acclimated culture, 6.25 mL was inoculated into 393.75 mL fresh Hypho media in a 2 liter flask and grown under the same conditions. Bacterial cells were harvested at mid log phase (OD600~0.13).
|
Growth protocol |
10 uL frozen culture were inoculated in 9.6 mL Hypho media supplemented with 15 mM of methanol. The culture was incubated at 30 °C with 225 rpm shaking for 4 days. 0.15 mL culture was then transferred to 9.45 mL of fresh Hypho media and grown under the same conditions for 4 days. From this acclimated culture, 6.25 mL was inoculated into 393.75 mL fresh Hypho media in a 2 liter flask and grown under the same conditions. Bacterial cells were harvested at mid log phase (OD600~0.13).
|
Extracted molecule |
total RNA |
Extraction protocol |
To obtain individual replicates for RNA extraction, samples from cultures were harvested under exponential growth phase and taken as rapidly as possible to limit any potential changes in transcript profiles. A culture volume of 50 mL was sampled directly from the 400 mL batch culture into a conical tube containing 1/10th the culture volume of ice-cold “Stop Solution” (5% water saturated phenol (pH 7.0), 95% absolute ethanol). Cultures were immediately harvested by centrifugation at 4500 g for 8 min at 4°C. Total RNA was extracted from M. extorquens AM1 pellets using the RNeasy Mini Kit (QIAGEN) according to the manufacture’s Yeast III protocol. Lysis was achieved by 3 min of bead-beating in the Mini-Beadbeater 8 (BioSpec Products, Bartlesville, OK) with 600 uL of 0.1-mm zirconium-silica beads (BioSpec Products). SUPERase•In (Ambion, Austin, TX) was added at a final concentration of 1 U/uL to prevent RNA degradation after the elution of RNA with RNase-free water. RNA concentration and purity were determined by using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). RNA integrity was assessed by using a Bioanalyzer (Agilent Technologies) as per manufacturer’s instructions. Residual DNA was removed from isolated RNA samples by treating 60 ug RNA with 36 units of RNasefree DNaseI (Ambion) in a 300-uL reaction volume for 1 h at 37°C. DNaseI-treated total RNA samples were purified by using the RNeasy Mini Kit (QIAGEN) according to the manufacture’s RNA cleanup protocol with a slight modification. Briefly, 1050 uL Buffer RLT and 750 uL ethanol were added to the 300 uL RNA samples and subsequently loaded onto an RNeasy mini column and centrifuged. The remaining RNA sample was successively loaded onto the same column and processed until the rest of the RNA sample was collected on the column. The column was washed three times with 700 uL buffer RW1, and then three times with 500 uL RPE buffer. DNaseI-treated total RNA was eluted with 30 uL of RNase-free water. RNA concentration, purity, and integrity were determined as above. The presence of DNA contamination was examined by PCR. DNaseI-treated total RNA (100 ng) was used as a template in a 50-uL reaction. PCR was done using Taq DNA polymerase (Promega, Madison, WI) with 40 cycles. Only RNA that did not result in a PCR product was used for cDNA synthesis and labeling.
|
Label |
Alexa Fluor 647
|
Label protocol |
The cDNA synthesis and labeling reactions were carried out by using an Amino Allyl cDNA Labeling Kit (Ambion) and Alexa Fluor 555 and Alexa Fluor 647 Reactive Dyes (Invitrogen Corporation, Carlsbad, CA) according to the manufacture’s manual with the following modifications: 5 ug DNaseI-treated total RNA and 4 ug random decamers were used for cDNA synthesis. Ethanol precipitated cDNA incorporating amino allyl dUTP was incubated overnight at 80°C and microcentrifuged for 45 min at 20,800 g at 4°C. Precipitated cDNA was coupled to either Alexa Fluor 555 or Alexa Fluor 647 reactive dye dissolved in 3 uL high-quality DMSO. Dye-labeled cDNA was purified using a QIAquick PCR Purification Kit (QIAGEN) following the instructions in the kit except for performing three washes instead of one. cDNA samples were eluted into 50 uL Nuclease-free water. The incorporation of dyes, the nucleic acid concentration, and the labeling efficiency of dye-labeled cDNA were measured and calculated as described above.
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|
|
Hybridization protocol |
Hybridization was performed according to the Agilent 60-mer oligo microarray processing protocol (Sure-Hyb chamber/SSPE wash with the Agilent Stabilization and Drying Solution) except that a microhybridization oven (Bellco Biotechnology, Vienland, NJ) was used. The appropriate amount of labeled cDNA to use for hybridizations was examined by generating a Ratio-Intensity (RI) plot. The condition in which the majority of the values of [1/2 log2(R*G)] showed 10 to 12 (1024 to 4096) was selected to strike a good balance between intensities that are too close to background and those that approach the limits of the dynamic range of the assay. Alexa Fluor 555- and Alexa Fluor 647-labeled cDNAs (40 pmol) were cohybridized. After hybridized slides had been washed and dried, they were kept in a nitrogen-purged SmartDesiccator (Terra Universal, Fullerton, CA) until scanning. Unused array slides were also kept under the nitrogen purged SmartDesiccator.
|
Scan protocol |
Microarray slides were sent to the Mogene (Saint Louis, MO) and scanned using an Agilent G2565AA microarray scanner. Scanned images were processed by Agilent Feature Extraction software Ver. 7.5.1 using the default settings for Agilent arrays, the Linear & Lowess method for normalization, and PMT settings of 10, 50, and 100 to obtain usable signal intensities from weak through strong signals. These results showed that for most of the probes, data from the 100 PMT setting were optimal. However, data from the 10-PMT setting were used for the signals (g and r mean) whose intensities were >50,000 at the 100-PMT setting.
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Description |
Strain CM1145 is an evolved isolate of strain CM702 after 600 generations of experimental evolution. Evolution experiments proceeded at 30 degrees C with 225 rpm shaking in 9.6 ml batch culture supplemented with 15 mM of methanol.Strain cm702 strain is distinguished from the wt by the following genetic characteristics: deletion of the mptG gene, carrying the the disrupted crtI502 allele, and expressing flhA and fghA genes from Paracoccus denitrificans.
|
Data processing |
The data in the VALUE column in the table were computed by taking the Log10 of the ratio of the lowess normalized Alexa_647 signal (rDyeNormSignal) divided by the lowess normalized Alex_555 signal (gDyeNormSignal) for each spot.
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Submission date |
Feb 17, 2009 |
Last update date |
Feb 24, 2009 |
Contact name |
HSIN-HUNG CHOU |
E-mail(s) |
hchou@fas.harvard.edu
|
Organization name |
Harvard University
|
Department |
Organismic and Evolutionary Biology
|
Lab |
Christopher Marx
|
Street address |
16 Divinity Avenue, Biolab 3079
|
City |
Cambridge |
State/province |
MA |
ZIP/Postal code |
02138 |
Country |
USA |
|
|
Platform ID |
GPL6262 |
Series (1) |
GSE14875 |
Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation |
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