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| Status |
Public on Apr 01, 2024 |
| Title |
Furfural |
| Sample type |
SRA |
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| Source name |
Escherichia coli
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
type: plasmids
treatment: PCR
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| Growth protocol |
Acetate, NaCl, furfural, isobutanol and high temperature were selected as stress screening conditions to verify the library tolerance in mineral medium (M9 medium) . M9 medium contained (per liter of deionized water) 1 g of NH4Cl, 0.5 g of NaCl, 4 g of Na2HPO4, and 3 g of KH2PO4. The following components were sterilized separately and then added to M9 medium (per liter [final volume] of medium): 2 mL of 1 M MgSO4, 1 mL of 0.1 M CaCl2, glucose or xylose, 10 mL of a trace element solution containing (per liter) 1 g of FeCl3, 0.18 g of ZnSO4· 7 H2O, 0.12 g of CuCl2· 2 H2O, 0.12 g of MnSO4· H2O, and 0.18 g of CoCl2· 6 H2O
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mutations on chromosomes were consistent with those carried by the CREATE plasmids. Firstly, the CREATE plasmids of tolerant strains were extracted separately. Following extraction, the CREATE plasmids were used as templates for PCR using Illumina-compatible primers designed with Golay barcodes . CREATE library fragments were amplified in 16 cycles of PCR. At the first PCR step, each experimental sample was ligated with a unique barcode used to track the mutations. After that, PCR products were confirmed by electrophoresis and purified using an Agarose Gel DNA Extraction Kit. Deep sequencing was performed according to the Illumina protocol for amplicon sequencing.
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.These libraries were divided into G1, G2, G3, G4 and G5 sub-groups according to different gene functions (such as active sites, DNA binding sites, and predicted functional sites) .
Illumina protocol for amplicon sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DNA fragment
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| Data processing |
Illumina protocol for amplicon sequencing
The editing cassette was used to quantify fitness associated with different mutations in the library. Fitness associated with all mutations in the library was calculated using log2-fold enrichment. Synonymous mutations of every homology arm were regard as a part of each corresponding library. Synonymous mutations were used to determine the standard deviation and mean value of the log2-fitness change of wild-type cells. Then, a mean ± 2σ cutoff (i.e., p=0.05 assuming a normal distribution) was used to estimate strongly enriched mutations in the selections. The confidence interval of 95% was used to calculate statistically significant differences between samples. Differences with p ≤ 0.05 were considered statistically significant.
Assembly: Escherichia coli str. K-12 substr. MG1655, complete genome.
Supplementary files format and content: 91 potential positive mutations selected by CREATE deep sequencing and fitness calculation
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| Submission date |
Apr 07, 2022 |
| Last update date |
Apr 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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| Platform ID |
GPL21117 |
| Series (1) |
| GSE200448 |
Deep sequencing and fitness calculation for plasmids carrying the CREATE cassette from the enriched tolerant strains under different pressures by using Illumina protocol |
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| Relations |
| BioSample |
SAMN27406440 |
| SRA |
SRX14779549 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6033849_F_Significant_Mutations.txt.gz |
2.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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