diff --git a/assays/WholeGenomeSequencing/isa.assay.xlsx b/assays/WholeGenomeSequencing/isa.assay.xlsx index 8b7965f413a8b1f6416d37abc8f5f771f6e458f5..0b091327a361c4ad8da281e7267bed1ddca4c750 100644 Binary files a/assays/WholeGenomeSequencing/isa.assay.xlsx and b/assays/WholeGenomeSequencing/isa.assay.xlsx differ diff --git a/assays/WholeGenomeSequencing/protocols/WholeGenomeSequencing.md b/assays/WholeGenomeSequencing/protocols/WholeGenomeSequencing.md index 9f2766c08b69d5b2c406b6e995b93cede8acecd4..c50e2df39eebb072cc66352086b263d4ba6fa3bf 100644 --- a/assays/WholeGenomeSequencing/protocols/WholeGenomeSequencing.md +++ b/assays/WholeGenomeSequencing/protocols/WholeGenomeSequencing.md @@ -1,16 +1 @@ -Overview of clones selected for whole genome sequencing: - -- pfkA 1: clone from ALE of ::PhrtB-pfkA on plates (first round) -- pfkA 2: clone from ALE of ::PhrtB-pfkA on plates (first round) -- pfkA 3: clone from ALE of ::PhrtB-pfkA on plates (second round) -- pfkA 4: clone from ALE of ::PhrtB-pfkA on plates (second round) -- pfkA 5: clone from ALE of ::PhrtB-pfkA on plates (second round) -- pfkA 6: clone from fALE of ::PhrtB-pfkA, liquid-automated -- pfkA 7: clone from fALE of ::PhrtB-pfkA, liquid-automated -- pfkA 8: clone from fALE of ::PhrtB-pfkA, liquid-automated -- pfkA control: parental ::PhrtB-pfkA strain, unevolved -- aceE 1: clone from ALE of ::PhrtB-aceE on plates (second round) -- aceE 2: clone from ALE of ::PhrtB-aceE on plates (second round) -- aceE 3: clone from ALE of ::PhrtB-aceE on plates (second round) -- aceE 4: clone from ALE of ::PhrtB-aceE on plates (second round) -- aceE 5: clone from ALE of ::PhrtB-aceE on plates (first round) \ No newline at end of file +Whole genome sequencing (WGS) of isolated C. glutamicum clones was performed using next generation sequencing (NGS). First, genomic DNA was prepared using NucleoSpin microbial DNA kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. Concentrations of the purified genomic DNA were measured using Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, United States) according to manufacturer’s instructions. The purified genomic DNA was used for the preparation for genome sequencing using NEBNext Ultra II DNA Library Kit for Illumina (New England BioLabs, Frankfurt am Main) and MiSeq Reagent Kit v2 (300-cycles) (Illumina, San Diego, CA, United States), according to manufacturer’s instructions. A MiSeq system (Illumina, San Diego, CA, United States) was used for sequencing. Data analysis and base calling were accomplished with the Illumina instrument software. FASTQ output files were analyzed for single nucleotide polymorphisms using BV-BRC web resources (3.34.11, https://www.bv-brc.org/) (Olson et al., 2023) via variation analysis and CLC Workbench 20.0.4 (https://digitalinsights.qiagen.com/) for structural variants and InDel analysis. diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index 37a4bbce81d3aea9aea527da7557a85ab91d7c98..20efc698a25a4b6d39099cc2b88f3a3f5496a3f7 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/ALECloneScreening/isa.study.xlsx b/studies/ALECloneScreening/isa.study.xlsx index 5c3e3f72c3284cf16d1d8b45a92508a963acb968..19440fcd675fa5238ac6a4409358b6987ca6539f 100644 Binary files a/studies/ALECloneScreening/isa.study.xlsx and b/studies/ALECloneScreening/isa.study.xlsx differ diff --git a/studies/ALECloneScreening/protocols/WholeGenomeSequencing.md b/studies/ALECloneScreening/protocols/WholeGenomeSequencing.md index 66d1594f658e0450b89e9e66583430b719ef5c51..243095cbb75c0c3a0f79784cd67305f19ab72399 100644 --- a/studies/ALECloneScreening/protocols/WholeGenomeSequencing.md +++ b/studies/ALECloneScreening/protocols/WholeGenomeSequencing.md @@ -1 +1,25 @@ -Whole genome sequencing (WGS) of isolated C. glutamicum clones was performed using next generation sequencing (NGS). First, genomic DNA was prepared using NucleoSpin microbial DNA kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. Concentrations of the purified genomic DNA were measured using Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, United States) according to manufacturer’s instructions. The purified genomic DNA was used for the preparation for genome sequencing using NEBNext Ultra II DNA Library Kit for Illumina (New England BioLabs, Frankfurt am Main) and MiSeq Reagent Kit v2 (300-cycles) (Illumina, San Diego, CA, United States), according to manufacturer’s instructions. A MiSeq system (Illumina, San Diego, CA, United States) was used for sequencing. Data analysis and base calling were accomplished with the Illumina instrument software. FASTQ output files were analyzed for single nucleotide polymorphisms using BV-BRC web resources (3.34.11, https://www.bv-brc.org/) (Olson et al., 2023) via variation analysis and CLC Workbench 20.0.4 (https://digitalinsights.qiagen.com/) for structural variants and InDel analysis. \ No newline at end of file +Selection on agar plates + +Pre-cultures of C. glutamicum ::PhrtB-pfkA and ::PhrtB-aceE each containing pJC1-PhrtB-eyfp were inoculated in 5 ml BHI supplemented with 25 µM kanamycin and incubated over night at 30°C, 170 rpm. On the following day, pre-cultures were diluted in 0.9% NaCl and the dilutions 10-3 to 10-5 were streaked out on CGXII agar plates supplemented with 2% glucose, 100 µM FeSO4 (iron excess) and kanamycin. Plates were incubated at 30 °C and pictures taken every 24 h. Clones were picked, covering diverse colony morphologies and fluorescence intensities were observed with a stereomicroscope (Nikon SMZ18 (λEx : 500/20, λEm : 535/30)). + +Automated f(luorescent)ALE for selection in liquid media + +In triplicates, pre-cultures of C. glutamicum ::PhrtB-pfkA containing pJC1-PhrtB-eyfp were inoculated in 1 ml complex medium BHI, and 20 µl were used to inoculate the first main culture well in the microbioreactor. The medium for main cultures in the microbioreactor ALE was CGXII containing 2 % glucose, 100 µM FeSO4 (i.e. iron excess) and kanamycin. Biomass and fluorescence of active cultures was monitored online, and employed to trigger automated inoculation of subsequent batches. The inoculation of all subsequent batches was triggered when >50 % of the currently active cultures reached a backscatter threshold of 120. New batches were prepared with 770 µl fresh medium, and 30 µl inoculum drawn from the culture with highest specific fluorescence. Cultures with lower specific fluorescence were not used for inoculation. Backscatter and fluorescence signals were smoothed with a crossvalidated smoothing spline (38) to improve signal stability. Completed batches were harvested to a cooled storage position. The three final batches were streaked on BHI plates for single clone selection. + + + +Overview of clones selected for whole genome sequencing: +- pfkA 1: clone from ALE of ::PhrtB-pfkA on plates (first round) +- pfkA 2: clone from ALE of ::PhrtB-pfkA on plates (first round) +- pfkA 3: clone from ALE of ::PhrtB-pfkA on plates (second round) +- pfkA 4: clone from ALE of ::PhrtB-pfkA on plates (second round) +- pfkA 5: clone from ALE of ::PhrtB-pfkA on plates (second round) +- pfkA 6: clone from fALE of ::PhrtB-pfkA, liquid-automated +- pfkA 7: clone from fALE of ::PhrtB-pfkA, liquid-automated +- pfkA 8: clone from fALE of ::PhrtB-pfkA, liquid-automated +- pfkA control: parental ::PhrtB-pfkA strain, unevolved +- aceE 1: clone from ALE of ::PhrtB-aceE on plates (second round) +- aceE 2: clone from ALE of ::PhrtB-aceE on plates (second round) +- aceE 3: clone from ALE of ::PhrtB-aceE on plates (second round) +- aceE 4: clone from ALE of ::PhrtB-aceE on plates (second round) +- aceE 5: clone from ALE of ::PhrtB-aceE on plates (first round) \ No newline at end of file