Project description:Metabolically-engineered Escherichia coli has been used previously to degrade the ubiquitous pollutant cis-1,2-dichloroethylene (cis-DCE), and the impact of the metabolic engineering was assessed by investigating the changes in the proteome. Here, genome-wide transcriptome analysis was performed to confirm that a strong heat shock and/or oxidative stress occurs during enhanced cis-DCE mineralization. Also, seven new stress proteins (YchH, YdeI, YodD, YodC, YgiW, YhcN, and YjaA) that were previously uncharacterized have been identified. Experiment Overall Design: Twelve planktonic RNA samples for the six sets of microarray experiments (set i was repeated) were prepared (Table 1 indicates the rationale of the whole-transcriptome studies): (i) cells of E. coli TG1 with TOM-Green/IsoILR1/GSHI* vs. no cloned genes contacted for 2 h with 1 mM cis-DCE to determine the effect of expressing eight genes (encoding TOM-Green/IsoILR1/GSHI*) on cis-DCE mineralization, (ii) cells of TG1 with TOM-Green/EchA F108L/I219L/C248I vs. TOM-Green contacted for 2 h with 1 mM cis-DCE to determine the effect of the evolved epoxide hydrolase on cis-DCE mineralization, (iii) cells (turbidity of 1.0) of E. coli BW25113 with 2 mM H2O2 vs. no H2O2 contacted for 10 min with shaking to determine gene expression in response to H2O2 stress since cis-DCE mineralization induced many oxidative stress genes (iv) cells (turbidity of 1.0) of the BW25113 ygiW strain with 2 mM H2O2 vs. no H2O2 contacted for 10 min to determine gene expression in response to H2O2 stress in the absence of YgiW, and (v) cells (turbidity of 1.0) of the BW25113 ychH strain with 2 mM H2O2 vs. no H2O2 contacted for 10 min to determine gene expression in response to H2O2 stress in the absence of YchH. Experiment Overall Design: For the cis-DCE degradation system, the exponentially-grown TG1 cells (Table 1) were contacted with 1 mM cis-DCE together with 0.5 mM IPTG and 5 mM succinate (as a carbon/energy source during the cis-DCE degradation) in 60 mL vials for 2 hr as performed previously in the proteomics study J. Proteome Res. 5: 1388 - 1397 (2006). After 2 hr contact with cis-DCE, samples were centrifuged, and the cell pellets were frozen immediately with dry ice and stored at -80°C. For the H2O2 stress system, overnight cultures of BW25113, BW25113 ygiW, and BW25113 ychH were re-grown to mid-log phase in LB (turbidity at 600 nm of 1), then the cultures were split into two cultures and 2 mM H2O2 and water (negative control) were added. After contacting for 10 min, samples were centrifuged and the cell pellets frozen immediately with dry ice and stored at -80°C. RNA was isolated from the biofilm cells as described previously using sonication and a bead beater Appl. Microbiol. Biotechnol. 64: 515-524 (2004).. Experiment Overall Design: For DNA microarray analysis, the E. coli Genechip antisense genome array (Affymetrix, P/N 900381, Santa Clara, CA) and the E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N 900551) were used to study the differential gene expression profile of the K12 cells; the E. coli Genechip antisense genome array contains probe sets for all 4290 open reading frames (ORF), rRNA, tRNA, and 1350 intergenic regions, and the E. coli GeneChip Genome 2.0 Array contains 10,208 probe sets for open reading frames, rRNA, tRNA, and intergenic regions for four E. coli strains: MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933. The same type of microarray was used for each binary comparison. cDNA synthesis, fragmentation, and hybridizations were as described previously J. Bacteriol. 188: 587-598 (2006). Hybridization was performed for 16 h, and the total cell intensity was scaled to an average value of 500. The probe array images were inspected for any image artifact. Background values, noise values, and scaling factors of both arrays were examined and were comparable. The intensities of polyadenosine RNA control were used to monitor the labeling process. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 11-15 probe pairs (p-value less than 0.05), these genes were discarded. Experiment Overall Design: Extract protocol: To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 50 seconds at the maximum speed in a mini bead beater (cat. no. 3110BX, Biospec). The total RNA was isolated by following the protocol of the RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase I (Qiagen). Experiment Overall Design: Label protocol: The total RNA samples were first converted into cDNA through a reverse transcription reaction with poly-A RNA controls spiked into the same reaction mixture to monitor the entire target labeling process. The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50-200 bp fragments, which was checked by running the fragmented cDNA on a 2% agarose gel. The fragmented cDNA was labeled at the 3’ termini by the Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP (Affymetrix, P/N 900181). Experiment Overall Design: Hybridization protocol: The biotin-labeled target was hybridized to the Affymetrix GeneChip E. coli array at 45C for 16 hour at 60 rpm using the Hybridization Oven 640 (Affymetrix), then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure. Experiment Overall Design: Scanning protocol: The microarray was scanned at 570 nm to get an image file by the GeneChip Scanner 3000 (Affymetrix). Using GeneChip® Operating Software, total cell intensity was scaled automatically in the software to an average value of 500.
2009-11-27 | E-GEOD-13698 | biostudies-arrayexpress
