|
| Status |
Public on Oct 01, 2024 |
| Title |
Glucose as carbon source |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control samples of the exponential growth phase of wild-type BL21(DE3)in cultures with glucose as a carbon source as control
|
| Organism |
Escherichia coli BL21(DE3) |
| Characteristics |
strain: BL21(DE3)
|
| Treatment protocol |
The strains were grown on mineral medium with glucose or xylose as carbon source
|
| Growth protocol |
The strains wild-type (BL21DE3)) and the xylose-selective strain (BL21(DE3) Δglk, ΔmanZ, ΔptsG, xylR::Kmr, lacZ::xylRC91A-Gmr) were grown on minimal medium M9 (pH 7) supplemented with 20 mM MOPS and 33 mM xylose or 28 mM glucose. The cultures were carried out in a 250 mL flask with 50 mL medium at 300 rpm and 37 °C until strains reached the middle exponential phase (~ 0.3-0.4 gDCW L-1)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 mL of middle exponential phase culture were mixed with 500 µL of RNAlaterTM (Thermo Fisher Scientific, Baltics, UAB), then gently mixed by inversion. The samples were centrifugated at 5,000 rpm for 5 minutes, the supernatant was discarded, and the pellet was frozen at -70 °C until further analysis. Total RNA was isolated using an RNAeasy Midi kit (cat. no. 75142, Qiagen, Hilden, Germany). Chromosomal DNA was removed with the turbo-DNA free kit (cat no. AM1907, Thermo Fisher Scientific, Baltics, UAB).
|
| Label |
Alexa555, Alexa647
|
| Label protocol |
10 ug of total RNA were used for cDNA synthesis, incorporating dUTP-Alexa555 or dUTP-Alexa647, employing the SuperScript Plus direct labeling kit (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
Experimental samples of the exponential growth phase of xylose-selective strain derived from BL21(DE3) in cultures with glucose as a carbon source
|
| Organism |
Escherichia coli BL21(DE3) |
| Characteristics |
strain: ES04 (BL21(DE3) Δglk, ΔmanZ, ΔptsG, xylR::Kmr, lacZ::xylRC91A-Gmr)
|
| Treatment protocol |
The strains were grown on mineral medium with glucose or xylose as carbon source
|
| Growth protocol |
The strains wild-type (BL21DE3)) and the xylose-selective strain (BL21(DE3) Δglk, ΔmanZ, ΔptsG, xylR::Kmr, lacZ::xylRC91A-Gmr) were grown on minimal medium M9 (pH 7) supplemented with 20 mM MOPS and 33 mM xylose or 28 mM glucose. The cultures were carried out in a 250 mL flask with 50 mL medium at 300 rpm and 37 °C until strains reached the middle exponential phase (~ 0.3-0.4 gDCW L-1)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 mL of middle exponential phase culture were mixed with 500 µL of RNAlaterTM (Thermo Fisher Scientific, Baltics, UAB), then gently mixed by inversion. The samples were centrifugated at 5,000 rpm for 5 minutes, the supernatant was discarded, and the pellet was frozen at -70 °C until further analysis. Total RNA was isolated using an RNAeasy Midi kit (cat. no. 75142, Qiagen, Hilden, Germany). Chromosomal DNA was removed with the turbo-DNA free kit (cat no. AM1907, Thermo Fisher Scientific, Baltics, UAB).
|
| Label |
Alexa555, Alexa647
|
| Label protocol |
10 ug of total RNA were used for cDNA synthesis, incorporating dUTP-Alexa555 or dUTP-Alexa647, employing the SuperScript Plus direct labeling kit (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Incorporation of fluorophore was analyzed by using the absorbance at 555 nm and 647 respectively. Equal quantities of labeled cDNA were hybridized using hybridization solution HybIT2 (TeleChem International INC). The arrays were incubated for 14 h at 42 °C, and then washed tree times with 1X SCC, 0.05 % SDS at room temperature.
|
| Scan protocol |
Acquisition and quantification of array images were performed in GenePix 4100-A reader with its accompanying software. For each spot, the mean density, mean background, and mean normalized signal values were calculated with the Array-Pro Analyzer software from Media Cybernetics.
|
| Description |
Analysis was performed using the experimental samples of the xylose-selective ES04 (BL21(DE3) Δglk, ΔmanZ, ΔptsG, xylR::Kmr, lacZ::xylRC91A-Gmr) strain compared with the control samples of wild-type BL21(DE3). Both strains were cultured using glucose as a carbon source.
|
| Data processing |
Microarray data analysis was performed as follows. The data obtained from the quantification of the images were analyzed with Genarise in SWAP mode. The free software genArise, was developed in the Computing Unit of Cellular Physiology Institute of UNAM (http://www.ifc.unam.mx/genarise/). GenArise carry out a number of transformations: background correction, lowess normalization, intensity filter, replicates analysis and selecting differentially expressed genes. The goal of genArise is to identify which of the genes show good evidence of being differentially expressed. The software identifies differential expressed genes by calculating an intensity-dependent z-score. Using a sliding window algorithm to calculate the mean and standard deviation within a window surrounding each data point, and define a z-score where z measures the number of standard deviations a data point is from the mean.
zi = (Ri – mean(R)) / sd(R) Where zi is the z-score for each element, Ri is the log-ratio for each element, and sd(R) is the standard deviation of the log-ratio. With this criterion, the elements with a z-score > 2 standard deviations would be the significantly differentially expressed genes.
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| |
|
| Submission date |
Sep 21, 2024 |
| Last update date |
Oct 01, 2024 |
| Contact name |
Alfredo Martínez |
| E-mail(s) |
Alfredo.martinez@ibt.unam.mx
|
| Organization name |
Instituto de Biotecnología, Universidad Nacional Autónoma de México
|
| Department |
Departamento de Ingeniería Celular y Biocatálisis
|
| Lab |
Consorcio de Ingeniería Metabólica y Biología Sintética de Microorganismos
|
| Street address |
Av. Universidad 2001, Col. Chamilpa
|
| City |
Cuernavaca |
| State/province |
Morelos |
| ZIP/Postal code |
62210 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL34929 |
| Series (1) |
| GSE277741 |
Transcriptomic response of the xylose-selective strain derived from E. coli BL21(DE3) growing on xylose or glucose |
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