diff --git a/assays/AbsorptionSpectraAndODMeasurements/isa.assay.xlsx b/assays/AbsorptionSpectraAndODMeasurements/isa.assay.xlsx index c7447e4a0df9b94b75fd07f2a82cc3d85b64d4d2..dfa20d8968ca0b72391682d571b53b7c6a3576c1 100644 Binary files a/assays/AbsorptionSpectraAndODMeasurements/isa.assay.xlsx and b/assays/AbsorptionSpectraAndODMeasurements/isa.assay.xlsx differ diff --git a/assays/DCWMeasurement/isa.assay.xlsx b/assays/DCWMeasurement/isa.assay.xlsx index d9e740f8d0e487b4df70fa2761ae7809d87f3041..7714e81c20dcfafff88e6ed39b5e09d3a782afad 100644 Binary files a/assays/DCWMeasurement/isa.assay.xlsx and b/assays/DCWMeasurement/isa.assay.xlsx differ diff --git a/assays/DCWMeasurement/protocols/DCWMeasurement.md b/assays/DCWMeasurement/protocols/DCWMeasurement.md index 6bf234ddf95f46eea023864f88b3fb15ffa9360a..177118a4a19ab429dc6f31e56b9acf3a6f342878 100644 --- a/assays/DCWMeasurement/protocols/DCWMeasurement.md +++ b/assays/DCWMeasurement/protocols/DCWMeasurement.md @@ -1,2 +1,3 @@ ## DCW measurement + Cell dry weight measurements were carried out by transferring the cell pellet of 2 ml of cyanobacterial culture to a pre-weighed PCR tube, which was incubated at 60°C for 20 h. The tube was weighed and the difference noted as the cell dry weight, with measurements carried out in triplicates. \ No newline at end of file diff --git a/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/isa.assay.xlsx b/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/isa.assay.xlsx index 6bb65d8f2ea30e85eb06bc8e73341555ad6867d5..700f7d74c699e6cd8092353906e7cdc34ff00351 100644 Binary files a/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/isa.assay.xlsx and b/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/isa.assay.xlsx differ diff --git a/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/protocols/GC-MSMeasurementsForTheQuantificationOfSqualene.md b/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/protocols/GC-MSMeasurementsForTheQuantificationOfSqualene.md index 115d371abca90c0169ee3ba8945e2b54e62aa1b4..27b0f7c0210833f9f81a48443f0ae4235c166ef9 100644 --- a/assays/GC-MSMeasurementsForTheQuantificationOfSqualene/protocols/GC-MSMeasurementsForTheQuantificationOfSqualene.md +++ 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). Squalene concentrations in the measured samples were calculated using a calibration curve with a squalene standard (Figure S2 (SI)). \ No newline at end of file diff --git a/assays/MetabolicModeling/isa.assay.xlsx b/assays/MetabolicModeling/isa.assay.xlsx index 878b6335f73c3984cb7f53951bc9f3d46b0f25a4..75fbcc60b0016062149f32a10567453391ebe5c7 100644 Binary files a/assays/MetabolicModeling/isa.assay.xlsx and b/assays/MetabolicModeling/isa.assay.xlsx differ diff --git a/assays/MetabolicModeling/protocols/MetabolicModelingForTheIdentificationOfAmplificationTargets.md b/assays/MetabolicModeling/protocols/MetabolicModelingForTheIdentificationOfAmplificationTargets.md index 7532025f780122f5f395998bc89db579ecb73b19..ba46f91f696bc0303a93ff5b3218f6694652093d 100644 --- a/assays/MetabolicModeling/protocols/MetabolicModelingForTheIdentificationOfAmplificationTargets.md +++ b/assays/MetabolicModeling/protocols/MetabolicModelingForTheIdentificationOfAmplificationTargets.md @@ -1,4 +1,5 @@ ## Metabolic modeling for the identification of amplification targets + All simulations are based on a genome-scale stoichiometric network model of *Synechocystis* published by Knoop and colleagues (Knoop and Steuer, 2015). 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. To visualize the flux distributions a simplified network was implemented with d3flux (v.0.2.7) (St. John, 2016), a d3.js based visualization tool for COBRApy models. \ No newline at end of file diff --git a/assays/PigmentQuantification/isa.assay.xlsx b/assays/PigmentQuantification/isa.assay.xlsx index 16531865e2bbb4211ee35dc94773ceca3c1a3ab6..205414e723dc21370243df04c056da4612ade4ad 100644 Binary files a/assays/PigmentQuantification/isa.assay.xlsx and b/assays/PigmentQuantification/isa.assay.xlsx differ diff --git a/assays/PigmentQuantification/protocols/PigmentQuantification.md b/assays/PigmentQuantification/protocols/PigmentQuantification.md index 685f42676f81f1a682bd2ae51523642bb49bd8a0..251dc24e287317e779c4296f2c267601bbdf6e64 100644 --- a/assays/PigmentQuantification/protocols/PigmentQuantification.md +++ b/assays/PigmentQuantification/protocols/PigmentQuantification.md @@ -1,4 +1,5 @@ ## Pigment quantification + Each culture (300 µL) was sampled after 72 hours at the end of the growth experiment. 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 Binary files a/assays/cPCR/isa.assay.xlsx and b/assays/cPCR/isa.assay.xlsx differ diff --git a/assays/qPCR/isa.assay.xlsx b/assays/qPCR/isa.assay.xlsx index 5f9b32da4914f6b1ad8e6c9d77bf6a96652ffee0..f68e337e5c7717add5060f0ea1ade5f720ea0c78 100644 Binary files a/assays/qPCR/isa.assay.xlsx and b/assays/qPCR/isa.assay.xlsx differ diff --git a/assays/qPCR/protocols/qRT-PCR.md b/assays/qPCR/protocols/qRT-PCR.md index 886784ac99488aab27c5b86efc9488f56c18ef69..c813b45056082d79bce5d9f7bf9d2a6eef26be10 100644 --- a/assays/qPCR/protocols/qRT-PCR.md +++ b/assays/qPCR/protocols/qRT-PCR.md @@ -1,2 +1,3 @@ ## Quantitative real-time PCR (qRT-PCR) + Cultures were sampled (0.5 ml) after 72 hours at the end of the growth experiment. 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)). Changes in gene expression as fold changes compared to the control were determined using the 2−ΔΔCT method, using rpoA as a housekeeping gene and the Δshc strain subjected to the same rhamnose concentration as a control. \ No newline at end of file diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index 72ab4b090510c2c8388cb0e296766191c9f1ec7a..fdaf9fdb6aac515b97f48b8714b09eddf085f085 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/Englund-2014/isa.study.xlsx b/studies/Englund-2014/isa.study.xlsx index 94c78835b03dc869dfaba57fd0d4eccada8d1e21..ab8902acd6c55e12f49f27285dc6d1c6d000c8d1 100644 Binary files a/studies/Englund-2014/isa.study.xlsx and b/studies/Englund-2014/isa.study.xlsx differ diff --git a/studies/KnoopAndSteuer-2015/isa.study.xlsx b/studies/KnoopAndSteuer-2015/isa.study.xlsx index fc93c1637f03774ff325e10c12fa2fd30ede346c..80f629eaf7080ddae6765a62d66dcb4187c21729 100644 Binary files a/studies/KnoopAndSteuer-2015/isa.study.xlsx and b/studies/KnoopAndSteuer-2015/isa.study.xlsx differ diff --git a/studies/MeasurementsOfSqualeneProduction/isa.study.xlsx b/studies/MeasurementsOfSqualeneProduction/isa.study.xlsx index d86086f434d5abca96dffff0ecea4baa1f542a62..522bafa87f35ef7b8daf6edee18bad3946fca6b0 100644 Binary files a/studies/MeasurementsOfSqualeneProduction/isa.study.xlsx and b/studies/MeasurementsOfSqualeneProduction/isa.study.xlsx differ diff --git a/studies/PlasmidAndStrainConstruction/isa.study.xlsx b/studies/PlasmidAndStrainConstruction/isa.study.xlsx index 9a001cb1ff6211afcf05b8b93467f7c64e102756..add116d522806561a03f88b2e6b9d513cab1c34d 100644 Binary files a/studies/PlasmidAndStrainConstruction/isa.study.xlsx and b/studies/PlasmidAndStrainConstruction/isa.study.xlsx differ diff --git a/studies/Synechocystis/isa.study.xlsx b/studies/Synechocystis/isa.study.xlsx index 75f8466ac578f82348fadfe265e50865a6cad79e..0eaab6e34adc7b597404a948dd8b83c7d2127528 100644 Binary files a/studies/Synechocystis/isa.study.xlsx and b/studies/Synechocystis/isa.study.xlsx differ