Comment[ArrayExpressAccession] E-MEXP-3264 Investigation Title A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum following a single n-butanol pulse Comment[Submitted Name] A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum following a single n-butanol pulse Comment[AEExperimentDisplayName] A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum following a single n-butanol pulse Comment[MIAMExpressLogin] hol_j Comment[MIAMExpressSubmissionID] 7625 Experiment Description In this study the transcriptional behavior of the natural solvent producing bacterium Clostridium acetobutylicum was investigated following n-butanol stress using DNA microarray analysis. Therefore, a phosphate-limited chemostat culture was established and n-butanol stress (0.9%) was added to acidogenic cells at pH 5.7. Experimental Design co-expression_design dose_response_design replicate_design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name COMPOUND DOSE Experimental Factor Type compound dose Person Last Name Janssen Person First Name Holger Person Email holger.janssen@uni-rostock.de Person Phone +493814986161 Person Affiliation Institue of Biological Sciences Person Address Divison of Microbiology, Albert-Einstein-Str. 3, Rostock, Germany, 18051, Germany Person Roles submitter Person Roles Term Source REF MGED Ontology Quality Control Type biological_replicate Comment[QualityControlDescription] A phosphate-limited chemostat culture was established at pH 5.7 which represents acidogenic growth of C. acetobutylicum (n=2). After reaching acidogenic steady-state growth a considerable amount of n-butanol (100 mM; ~0.9% [v/v]) was added as single pulse.
Microarray experiments were performed twice (n=2). Public Release Date 2011-12-31 Comment[ArrayExpressSubmissionDate] 2011-06-19 19:55:34 Publication Status not yet submitted Protocol Name P-MTAB-21491 P-MTAB-21492 P-MTAB-21493 P-MTAB-21494 P-MTAB-21495 P-MTAB-21496 P-MTAB-21497 Protocol Description For preparation of labeled cDNA 15 M-5g random hexamer primer were annealed in a volume of 10 M-5l to 25 M-5g of RNA by incubation at 70M-0C for 10 min. Then 1 mM dATP, dTTP, dGTP as well as 0.4 mM dCTP, 50 M-5M Cy3 or Cy5 labeled dCTP (GE-Healthcare, Munich, Germany), 10 mM DTT and 200 U SuperScript III reverse transcriptase (Invitrogen, Carlsbad, USA) where added and the labeling reactions with a total volume of 20 M-5l were incubated for 2-3 h at 42M-0C. RNA was removed from the formed heteroduplexes by addition 2 M-5l 2,5 M NaOH and incubation for 15 min at 37M-0C. Hydrolysis was stopped by addition of 10 M-5l 2M HEPES pH 7,0. Labeled cDNA was separated from the reaction mixture using GFX columns (GE-Healthcare, Munich, Germany) according to suppliersM-^R instructions with the only modification of washing the columns 4 times before elution of labeled cDNA. Incorporation of Cy-3 or Cy-5 was checked qualitatively by a spectrophotometric wavelength scan and was quantified by using molar extinction coefficients of 150.000 l mol-1 cm-1 (at 550nm) for Cy-3 and 250.000 l mol-1 cm-1 (at 650 nm) for Cy-5.
(Parameters: Amplification = none, Mass unit = Micro gram) Continuous cultures of Clostridium acetobutylicum were grown with 0.5 mM

phosphate at 37 M-0C and 200 rpm. The dilution rate was set to 0,075 / h.


(Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = phosphate limited minimal medium) Normalization was done by setting the arithmetic mean of the ratios equal to 1 (GenePix Pro 6.0 software). RNA isolation and purification:

1. incubate 15 ?l SDS (25 %) + 1200 ?l phenol in a 2 ml E-cup at 65M-0C for 5 min

2. suspend 2 ml cell pellet in cold AE-buffer (600 ?l)

3. add the suspended cells to hot SDS/phenol solution (see above) and vortex

4. incubation of the suspended cells in SDS/phenol for 10 min at 65M-0C (frequently vortex)

5. centrifuge 2 ml E-cup with suspended cells for 15 min at 4 M-0C and 5000 x g

6. carefully transfer the clear supernatant in a new 2 ml E-cup and add 100 ?l 2 M sodium acetate (pH 5.2) + 600 ?l phenol (vortex)

7. see above step 5

8. see above step 6

9. see above step 5

10. carefully transfer the clear supernatant in a new 2 ml E-cup

11. add 2.5 volumes of ice cold ethanol (96 %) and vortex

12. at least 2 h incubation at -20 M-0C

13. sedimentation of RNA by centrifugation (13000 rpm, 4 M-0C, 1 h)

14. discard supernatant and wash the pellet with ice cold ethanol (70%) (centrifugation: 10000 x g, 4 M-0C, 5 min)

15. discard supernatant and dry pellet under laminar flow (approx. 30 min)

16. suspend the pellet in 15 ?l TE buffer (pH 8)

17. storage the RNA at -70 M-0C



Dnase I treatment of RNA samples:

1. add to RNA sample (15 ?l): 180 ?l DNase I buffer (pH 7.5) + 5 ?l Dnase I (10 U/?l)

2. incubation at 37 M-0C for 30 min

3. add 15 ?l 2 M sodium acetate (pH 5.2) + 500 ?l phenol

4. centrifugation for 15 min, 4 M-0C, 5000 x g rpm

5. carefully transfer the clear supernatant in a new 2 ml E-cup

6. add 2.5 volumes of ice cold ethanol (96 %) and vortex

7. at least 2 h incubation at -20 M-0C

8. sedimentation of RNA by centrifugation (10000 x g, 4 M-0C, 1 h)

9. discard supernatant and wash the pellet with ice cold ethanol (70 %) (centrifugation: 10000 x g, 4 M-0C, 5 min)

10. discard supernatant and dry pellet under laminar flow (approx. 30 min)

11. suspend the pellet in 15 ?l TE buffer (pH 8)

12. storage the RNA at -70 M-0C



buffers and solutions:



AE buffer: sodium acetate (waterfree) 164 mg

EDTA 37 mg

A. dest ad 100 ml

(adjust of pH at 5.5 with acetic acid)



sodium acetate buffer: sodium acetate (waterfree) 16,41 g

A. dest ad 100 ml

(adjust of pH at 5.2 with acetic acid)



TE (10/1) buffer: 1 M Tris HCl (pH 8) 1200 ?l

0,2 M EDTA (pH 8) 600 ?l

A. dest ad 120 ml



Dnase I buffer: Tris 480 mg

MgCl2 120 mg

A. dest ad 100 ml

(adjust of pH at 5.5 with HCl)
(Parameters: Extracted product = total_RNA, Amplification = none) Scanning was done using a GenePix 4000B microarray scanner (Molecular

Devices, Canada) using the GenePix Pro 6.0 software.
(Parameters: Scanning hardware = GenePix 4000B [Axon Instruments], Scanning software = GenePix Pro [Axon Instruments]) For microarray analysis 2 ml cell pellets were collected by centrifugation at 4 M-0C and 10000 x g for 1 min. Afterwards pellets were frozen in liquid nitrogen and stored at -80 M-0C. The hybridization was done in Tom Freeman hybridization buffer for 15 hours at 45 M-0C with cDNA containing approximately 80 pmols of Cy-3 and Cy-5 in an automatic Lucidea slide processor (GE-Healthcare, Munich, Germany). Slides were washed using a program applying consecutive washes of two times with 1x SSC buffer containing 0,2% SDS and then with 0,1x SSC. At the end flushing hybridization chambers with isopropanol and evaporating the isopropanol with

air dried the slides


(Parameters: Chamber type = OTHER: automatic Lucidea slide processor (GE-Healthcare, Munich, Germany), Quantity of label target used = 25, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 45) Protocol Type labeling grow bioassay_data_transformation nucleic_acid_extraction image_acquisition specified_biomaterial_action hybridization Protocol Term Source REF MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology MGED Ontology Term Source Name MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php SDRF File E-MEXP-3264.sdrf.txt