PA1 delta cel
mixed sample
Protocols: Bacterial cells were cultivated in liquid minimal medium supplemented with 0.5% (v/v) methanol as the sole carbon source at 30 °C and 180 rpm until reaching mid- to late-exponential phase (OD₆₀₀ = 0.8–1.0). Cells were harvested by centrifugation at 5,000 × g for 10 minutes at 4 °C and washed once with cold PBS. Pellets were stored at –80 °C until further processing.   The experiment began with three independent starter cultures per strain: PA1 wild-type (labeled 1, 2, and 3) and PA1 Δcel (labeled 5, 9, and 10). Each culture was inoculated from a single colony previously grown on minimal medium agar plates supplemented with 0.5% methanol. Cultures were cultivated in liquid minimal medium with 0.5% methanol at 180 rpm and 30 °C. Once cultures reached exponential growth, aliquots were used to inoculate fresh flasks with incrementally increasing methanol concentrations. A total of 15 transfer steps were performed, including intermittent returns to 0.5% methanol to verify baseline growth. A glycerol stock of each inoculum was prepared prior to every transfer. Methanol concentrations were adjusted in the following sequence:  3%, 4%, 5%, 0.5%, 5%, 6%, 7%, 0.5%, 7%, 8%, 9%, 0.5%, 9%, 10%, and 11% (v/v). At the end of the experiment, cells from populations that grew at 11% methanol were used to prepare glycerol stocks and were also plated on minimal medium agar containing 0.5% methanol. Single colonies were isolated, regrown, and cryopreserved. Genomic DNA was extracted from these purified clones and subjected to whole-genome sequencing to identify single-nucleotide polymorphisms (SNPs) associated with enhanced methanol tolerance. Genomic DNA was isolated from M. extorquens strains using the NucleoBond® HMW DNA kit (Macherey-Nagel), following the manufacturer's protocol. An additional lysis step with lysozyme treatment (20 mg/mL, 60 min at 37°C) was included, befor treatment with Proteinase K and RNAseA. High-molecular-weight DNA was extracted using the anion exchange-based columns provided in the kit. The DNA was eluted, precipitated, washed and resuspended in H2O.   Genomic DNA was quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and 1 ng was used to prepare whole genome sequencing libraries with the Illumina Nextera XT kit according to the manufacturer's instructions. Library quality was assessed with the fragment analyzer employing the Agilent HS NGS Fragment kit. Absolute quantification of libraries was done by qPCR and the KAPA library quantification Kit (Roche) in a qTOWER3 G Thermal Cycler (Analytic Jena, Germany).
ENA first public
2026-07-24
INSDC center name
Philipps-Universität Marburg Center for Synthetic Microbiology (SYNMIKRO) AG Becker
INSDC status
public
Submitter Id
E-MTAB-17135:PA1 delta cel
broker name
ArrayExpress
collection date
not collected
genotype
PA1 delta cel
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
whole organism
sample name
E-MTAB-17135:PA1 delta cel
scientific_name
Methylorubrum extorquens
strain
Methylobacterium extorquens PA1 delta cel
component_organism
Methylorubrum extorquens
component_organism
Methylorubrum extorquens