From 63a69f1e0d1966d48cd028977f28480543907ee9 Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Wed, 14 Feb 2024 16:18:17 +0100 Subject: [PATCH] add high-level metadata to assays and studies --- .../isa.assay.xlsx | Bin 10307 -> 10308 bytes assays/DCWMeasurement/isa.assay.xlsx | Bin 9885 -> 9910 bytes .../protocols/DCWMeasurement.md | 1 + .../isa.assay.xlsx | Bin 12988 -> 13093 bytes ...surementsForTheQuantificationOfSqualene.md | 1 + assays/MetabolicModeling/isa.assay.xlsx | Bin 6898 -> 6930 bytes ...TheIdentificationOfAmplificationTargets.md | 1 + assays/PigmentQuantification/isa.assay.xlsx | Bin 6900 -> 6917 bytes .../protocols/PigmentQuantification.md | 1 + assays/cPCR/isa.assay.xlsx | Bin 9592 -> 9601 bytes assays/qPCR/isa.assay.xlsx | Bin 10421 -> 11256 bytes assays/qPCR/protocols/qRT-PCR.md | 1 + isa.investigation.xlsx | Bin 8570 -> 8571 bytes studies/Englund-2014/isa.study.xlsx | Bin 11311 -> 11312 bytes studies/KnoopAndSteuer-2015/isa.study.xlsx | Bin 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b/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/protocols/GC-MSMeasurementsForTheQuantificationOfSqualene.md @@ -1,4 +1,5 @@ ## GC-MS measurements for the quantification of squalene + Each culture (1.5 ml) was sampled after 72 hours at the end of the growth experiment. The sample was centrifuged at 14,000 g, for five minutes and 4°C. The supernatant was discarded and the pellet was frozen at -80°C until further processing. The pellet was extracted with 500 µL acetone, containing 25 µM β-sitosterol as internal standard, under agitation at 1000 rpm and 50°C for 10 min. 500 µL of 1 M NaCl was added and mixed by vortexing. After adding 250 µL hexane, the sample was vigorously mixed for 1 min and centrifuged for phase separation (1 min at 1,780 g and 4°C). The upper hexane phase was transferred into GC-MS vials and stored at -20°C until the analysis. GC-MS analysis was carried out using a Gerstel automatic liner exchange system with multipurpose sample MPS2 dual rail and two derivatization stations, used in conjunction with a Gerstel CIS cold injection system (Gerstel, Muehlheim, Germany). For every 10-12 samples, a fresh multibaffled liner was inserted. Chromatography was performed using the Agilent 7890B GC. Metabolites were separated on an Agilent HP-5MS column (30ml x 0.25mm), the oven temperature was ramped with 12.5 °C/min from 70 °C (initial temp for 2 min) to 320 °C (final temp hold 5 min). Metabolites were ionized and fragmented in an EI source (70V, 200 °C source temp) and detected using 7200 accurate mass Q-TOF GC-MS from Agilent Technologies. Data analysis was performed using Agilent MassHunter Quantitative Analysis B.09.00. Peaks were identified using already available EI-MS fragmentation data. Peaks were identified using characteristic fragment ions (Bhatia et al., 2013) and retention times of standards (Squalene: mass/charge (m/z) = 81.07, retention time (RT) = 9.5 min; β-sitosterol: m/z = 107.09, RT = 13.6 min). 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A modified, extended version was used, kindly provided by Ralf Steuer. All flux distributions have been calculated with constraint-based flux analysis using COBRApy (v.0.25.0) (Ebrahim et al., 2013). To simulate phototrophic growth, different constraints were applied to the model of Synechocystis (see Table S5 (SI)). FSEOF (Choi et al., 2010) was used to find amplification targets by simulating the transition from a wildtype to a production phenotype. All isoreactions were excluded for the transition experiments (Knoop and Steuer, 2015). The initial fluxes of all reactions were calculated by using the objective function to maximize the growth rate. Then, the theoretical maximum squalene production rate was calculated by setting the objective function as maximizing squalene flux. Subsequently, under constant light flux, the product formation flux rate was stepwise increased from 0% to 67% of the maximum achievable rate, while the growth rate was maximized. Only targets for which the overall mean flux rate from maximum biomass synthesis to maximum product synthesis increases were chosen. Additionally, only reactions that did not change flux direction during transition were considered. To confirm the results, flux variability analysis was performed for the selected targets, by stepwise increasing squalene flux from 0% to 67% of the maximum rate and subsequently maximizing biomass synthesis. For each simulation step, the variability of all selected targets was determined. 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The sample was centrifuged at 14,000 g for 5 minutes and 4°C. The supernatant was discarded and the pellet was resuspended in 100 μl water. The samples were frozen at -80°C until further processing. 900 μl of 100% methanol were added to the sample and the sample was mixed by vortexing. After incubation in the dark under gentle agitation for 1 h at 4°C the sample was centrifuged at 14,000 g for 5 minutes. The supernatant was transferred into a cuvette and an absorbance spectrum was measured from 400 nm to 750 nm. The absorbance spectra were divided by the OD750 or CDW and the amount of chlorophyll a in the sample was quantified by the absorbance maximum of chlorophyll a at 665 nm (A665nm) using following equation (Lichtenthaler and Buschmann 2001): *Chlorophyll content[μg/ml]=12.66 μg/ml * A665 nm* diff --git a/assays/cPCR/isa.assay.xlsx b/assays/cPCR/isa.assay.xlsx index 9d0a7030aa4654ffb5318498cc846aa807de695f..600018ee5c5f5b57233bff264af43dbac67f5528 100644 GIT binary patch delta 3313 zcmZWrc_5Ts7k@@ImM|g97>1d#WLI{QH`_~=P|3cO-7ttqX)0S9%kX5$u7xmJqb5tD zMbua#G)fpt$x<lB_Koqr@7Mc%_m6Y#J?A{<ocr8!@A<79yK2T|WwvWK2LJ$UfLf$w zHCGm77lVWMA*gL$`vwBr5qx4nG8J1a5=@2EE~y>G@!c}q3`;v#eP!hvEC4XV4*tOk z2MczK;luKNeiP;+u^88b^=r!wnr5{6!hTq4$dMJzZ);VH<x>?&KlKr%^lL5hsMKk4 zna5Lw_R2LXfj=S9GwOvyKjB&<n?Xg#@l~)lrI@GHm~GnPsIY&0m<Zz2_2gT(gbh<} zIebaB=Ui4-j}{<>XoLlrgC0EL=09mBb~7o;_><0Nit0D!wR@--LqdkV{D^9Zvb4i1 zy=yaBmxs9oox*mZksflhgy^*U<1>|17vG=VWBz(=x!?Jyd*&DNXb$jGRQT8?KYcT= 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The pellet was processed for RNA extraction using the PGTX method (dx.doi.org/10.17504/protocols.io.jm3ck8n, Pinto et al., 2009). The remaining DNA in the extracted RNA was removed by DNase digestion using the TURBO DNA-free™ (ThermoFischer) kit according to the manufacturer’s instructions. Extracted RNAs (250 ng) were used in a reverse transcriptase reaction using the RevertAid First Strand cDNA Synthesis Kit (ThermoFischer) according to the manufacturer’s instructions. The resulting cDNA was diluted 1:20. For performing qPCR, the DyNAmo ColorFlash SYBR Green qPCR Kit was used according to the manufacturer’s instructions. Primers for sqs, dxs and the housekeeping gene rpoA are shown in Table S2 (SI). Primer efficiencies were tested before performing qRT-PCR and were deemed sufficient to yield quantitative information (Figure S3; Table S3 (SI)). 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