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| Status |
Public on Mar 01, 2024 |
| Title |
Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and CRP-E182D Transcriptomes |
| Organism |
Escherichia coli |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Strain engineering for industrial production requires the improvement of tolerance to multiple unfavorable conditions. Here, we report using global regulator libraries based on the CRISPR-enabled trackable genome engineering (CREATE) method to engineer tolerance against multiple inhibitors in Escherichia coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target 34,340 mutations across 23 global regulators. A total of 69 specific mutations that respectively conferred tolerance to acetate, NaCl, furfural, and high temperature were isolated, confirmed, and evaluated. Among them, 32 novel reconstructed mutations exhibited better tolerance to the corresponding inhibitors, and the most dramatic mutation CRP-E182D conferred high cross-tolerance to acetate, NaCl and isobutanol. To further investigate the effects of this mutation in the CRP on acetate tolerance, whole-transcriptome sequencing (RNA-Seq) analysis was performed.
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| Overall design |
Mutant strain CRP-E182D was grown in the presence of 20 g/L sodium acetate and collected for transcriptome analysis. The wild-type strain MG1655 grown with 20 g/L sodium acetate was used as control. Each strain was grown in three independent replicates. RNA sequencing of 6 samples was performed using an Illumina HiSeq platform. The samples were collected during the mid-logarithmic phase, quickly frozen in liquid nitrogen and stored at -80 °C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The quantity and quality of the isolated RNA were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), Nano Drop (Thermo Fisher Scientific Inc.) and 1% agarose gel electrophoresis. The following library preparation required 1μg of total RNA with an RNA Integrity Number (RIN) value above 6.5. The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England BioLabs, Ipswich, MA, USA) was used to construct next-generation sequencing libraries. The libraries sequencing was carried out on an Illumina HiSeq platform using a 2×150 paired-end (PE) configuration. The sequences were processed and analyzed by GENEWIZ (Suzhou, China).
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| Contributor(s) |
Song X, Zheng Y, Li S |
| Citation missing |
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| Submission date |
Mar 25, 2022 |
| Last update date |
Mar 01, 2024 |
| Contact name |
Yangyang Zheng |
| E-mail(s) |
2018207233@tju.edu.cn
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| Organization name |
Tianjin University
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| Street address |
135 Yaguan Road, Jinnan District, Tianjin
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| City |
Tianjin |
| ZIP/Postal code |
300350 |
| Country |
China |
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| Platforms (1) |
| GPL14548 |
Illumina HiSeq 2000 (Escherichia coli) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA819865 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE199389_All.fpkm_anno.xlsx |
793.5 Kb |
(ftp)(http) |
XLSX |
| GSE199389_CK-2-VS-UV-1_Pathway_Enrichment.xlsx |
10.2 Kb |
(ftp)(http) |
XLSX |
| GSE199389_Group_CK-2-VS-UV-1_DE_anno.txt.gz |
422.5 Kb |
(ftp)(http) |
TXT |
| GSE199389_Group_CK-2-VS-UV-1_DE_significant_anno.txt.gz |
41.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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