induced by: pET28a-DsFbFP M49I treatment: plus 15min blue light
Treatment protocol
see growth protocol
Growth protocol
To analyze differences in global oxidative stress response in E. coli that were induced by Pp2FbFP and DsFbFP M49I, a DNA microarray-based analysis was conducted. E. coli cells expressing the respective photosensitizers were cultivated in LB media, supplemented with 50 μg ml-1 kanamycin (25 ml culture volume in 500 ml Erlenmeyer flasks, start OD580 = 0.05). The cultures were incubated at 37 °C and under continuous shaking (130 rpm) in the dark. LOV-FP expression was induced by addition of 0.4 mM IPTG during the logarithmic growth phase (~OD580 = 0.7). Three hours after induction, cultures were divided into two test cultures with a volume of 25 ml in 500 ml Erlenmeyer flaks and an OD580 of 0.5. For light treatment one of these cultures was placed between two blue-light emitting LED-panels (λ = 462 nm, 100 mW cm-2, Insta Elektro, Lüdenscheid, Germany, panel distance: 30 cm), whereas the second culture was kept in the dark. To ensure optimal oxygen supply of cells during irradiation process, cultures were supplemented with stirring bars and placed on magnetic stirrers. Since the selectivity and efficiency of light-driven ROS formation strongly vary between those proteins, we first evaluated different exposure times (5, 10, 15 and 30 minutes) for full induction of LOV-FP-mediated stress-response in E.coli. To this end, samples containing an OD580 of 3 were harvested after light treatment by centrifugation and the cell pellets were shock frozen in liquid nitrogen and subsequently stored at -80°C. RNA isolation, cDNA synthesis, microarray hybridization and data analyses were performed as described before92 except the utilization of DNA-microarrays carrying probes for the genes of E. coli MG1655 strain. Detailed transcriptome analyses of light-induced stress-response based on independent biological triplicates were finally carried out under best illumination conditions (i.e. Pp2FbFP: 5 min, DsFbFP M49I: 15 min) as described above.
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed with the RNeasy Kit from Qiagen as described (Hanke T., Nöh K., Noack S., Polen T., Bringer S., Sahm H., Wiechert W., Bott M. (2013) Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H. Appl Environ Microbiol 79: 2336-48; doi: 10.1128/AEM.03414-12)
Label
Cy5
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Neusser T., Polen T., Geissen R., Wagner R. (2010) Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising reduction in the expression of the translation machinery. BMC Genomics 11: 165; doi: 10.1186/1471-2164-11-165
induced by: pET28a-DsFbFP M49I treatment: no blue light
Treatment protocol
see growth protocol
Growth protocol
To analyze differences in global oxidative stress response in E. coli that were induced by Pp2FbFP and DsFbFP M49I, a DNA microarray-based analysis was conducted. E. coli cells expressing the respective photosensitizers were cultivated in LB media, supplemented with 50 μg ml-1 kanamycin (25 ml culture volume in 500 ml Erlenmeyer flasks, start OD580 = 0.05). The cultures were incubated at 37 °C and under continuous shaking (130 rpm) in the dark. LOV-FP expression was induced by addition of 0.4 mM IPTG during the logarithmic growth phase (~OD580 = 0.7). Three hours after induction, cultures were divided into two test cultures with a volume of 25 ml in 500 ml Erlenmeyer flaks and an OD580 of 0.5. For light treatment one of these cultures was placed between two blue-light emitting LED-panels (λ = 462 nm, 100 mW cm-2, Insta Elektro, Lüdenscheid, Germany, panel distance: 30 cm), whereas the second culture was kept in the dark. To ensure optimal oxygen supply of cells during irradiation process, cultures were supplemented with stirring bars and placed on magnetic stirrers. Since the selectivity and efficiency of light-driven ROS formation strongly vary between those proteins, we first evaluated different exposure times (5, 10, 15 and 30 minutes) for full induction of LOV-FP-mediated stress-response in E.coli. To this end, samples containing an OD580 of 3 were harvested after light treatment by centrifugation and the cell pellets were shock frozen in liquid nitrogen and subsequently stored at -80°C. RNA isolation, cDNA synthesis, microarray hybridization and data analyses were performed as described before92 except the utilization of DNA-microarrays carrying probes for the genes of E. coli MG1655 strain. Detailed transcriptome analyses of light-induced stress-response based on independent biological triplicates were finally carried out under best illumination conditions (i.e. Pp2FbFP: 5 min, DsFbFP M49I: 15 min) as described above.
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed with the RNeasy Kit from Qiagen as described (Hanke T., Nöh K., Noack S., Polen T., Bringer S., Sahm H., Wiechert W., Bott M. (2013) Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H. Appl Environ Microbiol 79: 2336-48; doi: 10.1128/AEM.03414-12)
Label
Cy3
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Neusser T., Polen T., Geissen R., Wagner R. (2010) Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising reduction in the expression of the translation machinery. BMC Genomics 11: 165; doi: 10.1186/1471-2164-11-165
Hybridization protocol
Purified cDNA samples to be compared were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent’s Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
Scan protocol
Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 6.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0).
Data processing
For calculation and normalization of ratios reflecting the relative mRNA levels (Ratio of Medians, GenePix Pro), GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).
