E. coli K-12 MC4100 Grown on M9 glycerol medium at 23˚C for ~10 generations to exponential phase (~50 hours)
Biomaterial provider
CA White-Ziegler
Growth protocol
Bacterial cultures were inoculated and grown as described previously (1,2). An inoculum from a 37˚C grown single colony was used to initiate parallel, colony matched 37˚C and 23˚C grown cultures. The cells from these cultures were harvested at equivalent optical densities after approximately 9-11 generations growth in early-mid exponential phase (OD600= 0.2-0.6). Cell pellets were subsequently frozen in liquid nitrogen and stored at -80ºC for RNA isolation. 1.) White-Ziegler, C. A., A. M. Black, S. H. Eliades, S. Young, and K. Porter. 2002. The N-acetyltransferase RimJ responds to environmental stimuli to repress pap fimbrial transcription in Escherichia coli. J. Bacteriol. 184:4334-4342. 2.)White-Ziegler, C. A., A. Villapakkam, K. Ronaszeki, and S. D. Young. 2000. H-NS controls pap and daa fimbrial transcription Escherichia coli in response to multiple environmental cues. J. Bacteriol. 182:6391-6400.
Extracted molecule
total RNA
Extraction protocol
For microarray analyses, RNA was isolated by phenol: chloroform extraction as described previously (1) with the modifications that the RNA was subjected to a second DNase digestion in solution and cleaned using a RNeasy column per manufacturers directions (Qiagen). 1.)White-Ziegler, C. A., M. L. Angus Hill, B. A. Braaten, M. W. van der Woude, and D. A. Low. 1998. Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor. Mol Microbiol 28:1121-37.
Label
Cy5 (635)
Label protocol
Synthesis, labeling of cDNA with Cy3/Cy5, and hybridization was performed using the 3DNA Array 350 RP Expression Array Detection Kit according to the manufacturer’s protocol (Genisphere). cDNA for each condition was prepared from 2 μg total RNA. cDNA from both temperatures (37ºC and 23ºC) was cohybridized to slides containing full-length PCR products from all 4,290 annotated open reading frames (ORFs) in E. coli MG1655. 1.) White-Ziegler, C. A., A. J. Malhowski, and S. D. Young. Human body temperature (37˚C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12. Journal of Bacteriology 2007. Vol. 189(15):5429-40. Epub 2007 May 25.
E. coli K-12 MC4100 Grown on M9 glycerol medium at 37˚C for ~10 generations to exponential phase (~14 hours)
Biomaterial provider
CA White-Ziegler
Growth protocol
Bacterial cultures were inoculated and grown as described previously (1,2). An inoculum from a 37˚C grown single colony was used to initiate parallel, colony matched 37˚C and 23˚C grown cultures. The cells from these cultures were harvested at equivalent optical densities after approximately 9-11 generations growth in early-mid exponential phase (OD600= 0.2-0.6). Cell pellets were subsequently frozen in liquid nitrogen and stored at -80ºC for RNA isolation. 1.) White-Ziegler, C. A., A. M. Black, S. H. Eliades, S. Young, and K. Porter. 2002. The N-acetyltransferase RimJ responds to environmental stimuli to repress pap fimbrial transcription in Escherichia coli. J. Bacteriol. 184:4334-4342. 2.)White-Ziegler, C. A., A. Villapakkam, K. Ronaszeki, and S. D. Young. 2000. H-NS controls pap and daa fimbrial transcription Escherichia coli in response to multiple environmental cues. J. Bacteriol. 182:6391-6400.
Extracted molecule
total RNA
Extraction protocol
For microarray analyses, RNA was isolated by phenol: chloroform extraction as described previously (1) with the modifications that the RNA was subjected to a second DNase digestion in solution and cleaned using a RNeasy column per manufacturers directions (Qiagen). 1.)White-Ziegler, C. A., M. L. Angus Hill, B. A. Braaten, M. W. van der Woude, and D. A. Low. 1998. Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor. Mol Microbiol 28:1121-37.
Label
Cy3 (532)
Label protocol
Synthesis, labeling of cDNA with Cy3/Cy5, and hybridization was performed using the 3DNA Array 350 RP Expression Array Detection Kit according to the manufacturer’s protocol (Genisphere). cDNA for each condition was prepared from 2 μg total RNA. cDNA from both temperatures (37ºC and 23ºC) was cohybridized to slides containing full-length PCR products from all 4,290 annotated open reading frames (ORFs) in E. coli MG1655. 1.) White-Ziegler, C. A., A. J. Malhowski, and S. D. Young. Human body temperature (37˚C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12. Journal of Bacteriology 2007. Vol. 189(15):5429-40. Epub 2007 May 25.
Hybridization protocol
Hybridization was completed as described previously (1). The hybridization solution (10 μl of cDNA mixture, 1 μl of denatured rat Hybloc DNA (1 μg/μl), 20 μl of 2X SDS buffer, 2 μl of dT blocker, and 7 μl of nuclease-free water) was placed over a prewarmed microarray slide and covered with a Corning 22x40 mm coverslip. The slide was then sealed in a hybridization chamber and submerged for 3 days in a 60˚C water bath. Microarray slides were washed for 10 minutes in 2X SSC, 0.2% SDS at 37ºC, 10 minutes in 2X SSC at room temperature, and 10 minutes in 0.2X SSC at room temperature. Slides were dried by centrifugation at 10,000 rpm for two minutes. For the second hybridization to allow the 3DNA capture reagent to bind to the complementary Cy3 or Cy5 specific 3DNA capture sequences ligated to the cDNAs, each microarray received 42 μl of hybridization solution (2.5 μl of Cy3 capture reagent, 2.5 μl of Cy5 capture reagent, 21 μl of 2X SDS buffer, 0.5 μl anti-fade reagent, and 15.5 μl of nuclease-free water) and hybridized as before via submersion for 3-4 hours in a 61ºC water bath. The slides were washed, dried , and kept in the dark until they were scanned and analyzed. 1.) White-Ziegler, C. A., A. J. Malhowski, and S. D. Young. Human body temperature (37˚C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12. Journal of Bacteriology 2007. Vol. 189(15):5429-40. Epub 2007 May 25.
Scan protocol
Five slides were used in the analysis with cDNAs, representing three independent growth experiments and two technical replicates (1). Hybridized slides were scanned using GenePix Pro 4.1 software (Axon Instruments, Inc.). Photomultiplier tube (PMT) voltage was altered for the initial scanning to ensure proper channel balance and decrease background. Any gene feature that had a signal-to-background ratio < 2.0 was rejected and not further analyzed. The ratio of medians values were used for further analysis. 1.) White-Ziegler, C. A., A. J. Malhowski, and S. D. Young. Human body temperature (37˚C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12. Journal of Bacteriology 2007. Vol. 189(15):5429-40. Epub 2007 May 25.
Description
This slide is a technical replicate of Rep3 in which the dyes have been flipped.
Data processing
Photomultiplier tube (PMT) voltage was altered for the initial scanning to ensure proper channel balance and decrease background. Any gene feature that had a signal-to-background ratio < 2.0 was rejected and not further analyzed. The ratio of medians values were used for further analysis.
