diff --git a/.gitattributes b/.gitattributes new file mode 100644 index 0000000000000000000000000000000000000000..97e6e92d510c5f6a4542778fa02543aaae1dd365 --- /dev/null +++ b/.gitattributes @@ -0,0 +1,2 @@ +publication/Data[[:space:]]Sheet[[:space:]]1.PDF filter=lfs diff=lfs merge=lfs -text +publication/fpls-14-1024981.pdf filter=lfs diff=lfs merge=lfs -text diff --git a/assays/measurements and quantification/README.md b/assays/measurements and quantification/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/measurements and quantification/dataset/.gitkeep b/assays/measurements and quantification/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/measurements and quantification/isa.assay.xlsx b/assays/measurements and quantification/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..cceb9bc4f273d8597c8e695a82cea1a7801b7e84 Binary files /dev/null and b/assays/measurements and quantification/isa.assay.xlsx differ diff --git a/assays/measurements and quantification/protocols/.gitkeep b/assays/measurements and quantification/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/measurements and quantification/protocols/Biomass measurements (DCW, OD, spectra).md b/assays/measurements and quantification/protocols/Biomass measurements (DCW, OD, spectra).md new file mode 100644 index 0000000000000000000000000000000000000000..df136c9d4ec45786f604b883d838310e64c2d4e9 --- /dev/null +++ b/assays/measurements and quantification/protocols/Biomass measurements (DCW, OD, spectra).md @@ -0,0 +1,4 @@ +## Biomass measurements (DCW, OD, spectra) +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. + +Absorption spectra and OD measurements were carried out in 1 ml polystyrene cuvettes in a SPECORD 200 Plus Spectrophotometer (Analytik Jena) with BG11 as a blank and as a reference sample. Samples were diluted with BG11 to be within an absorption range of 0.1 to 1.0 to ensure accurate measurements. Cell densities for *Synechocystis* were measured at 750 nm. \ No newline at end of file diff --git a/assays/measurements and quantification/protocols/GC-MS measurements for the quantification of squalene.md b/assays/measurements and quantification/protocols/GC-MS measurements for the quantification of squalene.md new file mode 100644 index 0000000000000000000000000000000000000000..115d371abca90c0169ee3ba8945e2b54e62aa1b4 --- /dev/null +++ b/assays/measurements and quantification/protocols/GC-MS measurements for the quantification of squalene.md @@ -0,0 +1,4 @@ +## 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/measurements and quantification/protocols/Pigment quantification.md b/assays/measurements and quantification/protocols/Pigment quantification.md new file mode 100644 index 0000000000000000000000000000000000000000..685f42676f81f1a682bd2ae51523642bb49bd8a0 --- /dev/null +++ b/assays/measurements and quantification/protocols/Pigment quantification.md @@ -0,0 +1,8 @@ +## 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* + +The amount of carotenoids in the sample was quantified by the absorbance maximum of the sum of carotenoids at 470 nm (A470nm) and a correction term considering absorbance of chlorophyll a at 470 nm (c(Chl a): concentration of chlorophyll a in the sample) using following Equation (Lichtenthaler and Buschmann 2001): + +*Carotenoid content[mg/ml]=(1000 μg/ml * A470 nm−1.91 * c(Chl))/225* \ No newline at end of file diff --git a/assays/measurements and quantification/protocols/Quantitative real-time PCR (qRT-PCR).md b/assays/measurements and quantification/protocols/Quantitative real-time PCR (qRT-PCR).md new file mode 100644 index 0000000000000000000000000000000000000000..886784ac99488aab27c5b86efc9488f56c18ef69 --- /dev/null +++ b/assays/measurements and quantification/protocols/Quantitative real-time PCR (qRT-PCR).md @@ -0,0 +1,2 @@ +## 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/assays/metabolic modeling/README.md b/assays/metabolic modeling/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/metabolic modeling/dataset/.gitkeep b/assays/metabolic modeling/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/metabolic modeling/dataset/Table 2.XLSX b/assays/metabolic modeling/dataset/Table 2.XLSX new file mode 100644 index 0000000000000000000000000000000000000000..6d99fd94cbfb68d0621fd60b972a0105a6a585b5 Binary files /dev/null and b/assays/metabolic modeling/dataset/Table 2.XLSX differ diff --git a/assays/metabolic modeling/dataset/Table 3.XLSX b/assays/metabolic modeling/dataset/Table 3.XLSX new file mode 100644 index 0000000000000000000000000000000000000000..4e9e7a6f5b24d881863f76c70a4ee1f50b4c395b Binary files /dev/null and b/assays/metabolic modeling/dataset/Table 3.XLSX differ diff --git a/assays/metabolic modeling/isa.assay.xlsx b/assays/metabolic modeling/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..5c1458590f880182bcc14fdff8fca620214aa65a Binary files /dev/null and b/assays/metabolic modeling/isa.assay.xlsx differ diff --git a/assays/metabolic modeling/protocols/.gitkeep b/assays/metabolic modeling/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/metabolic modeling/protocols/Metabolic modeling for the identification of amplification targets.md b/assays/metabolic modeling/protocols/Metabolic modeling for the identification of amplification targets.md new file mode 100644 index 0000000000000000000000000000000000000000..7532025f780122f5f395998bc89db579ecb73b19 --- /dev/null +++ b/assays/metabolic modeling/protocols/Metabolic modeling for the identification of amplification targets.md @@ -0,0 +1,4 @@ +## 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/metabolic modeling/protocols/Table 1.XLSX b/assays/metabolic modeling/protocols/Table 1.XLSX new file mode 100644 index 0000000000000000000000000000000000000000..dc338db42577dc969af0b61bc5b6433f78598e89 Binary files /dev/null and b/assays/metabolic modeling/protocols/Table 1.XLSX differ diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index f78f6a5104f5a161661a01c8097145807c954be9..10bc9219caa187b7ce10414f99c4efa11b0b119e 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/publication/Data Sheet 1.PDF b/publication/Data Sheet 1.PDF new file mode 100644 index 0000000000000000000000000000000000000000..552aa3992652dfeeba825ed1511de94150c4d2ef --- /dev/null +++ b/publication/Data Sheet 1.PDF @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:62d03b2ee6206e9e97ae3b6460af84f35995eee29514b03d8c33b04c857d109d +size 1057497 diff --git a/publication/fpls-14-1024981.pdf b/publication/fpls-14-1024981.pdf new file mode 100644 index 0000000000000000000000000000000000000000..2aa4673a88a4799f757c3d40c073f9e0c446bb79 --- /dev/null +++ b/publication/fpls-14-1024981.pdf @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:366c9d43c36319b2ca0248836ec2faa2b9ee0e03bfd1e4afe9e605bf8889b9ff +size 3265718 diff --git a/studies/Synechocystis/README.md b/studies/Synechocystis/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Synechocystis/isa.study.xlsx b/studies/Synechocystis/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..3aeed844765339c37fd19617c9ab8f70f3ca6679 Binary files /dev/null and b/studies/Synechocystis/isa.study.xlsx differ diff --git a/studies/Synechocystis/protocols/.gitkeep b/studies/Synechocystis/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Synechocystis/protocols/Culture conditions.md b/studies/Synechocystis/protocols/Culture conditions.md new file mode 100644 index 0000000000000000000000000000000000000000..257fbb925ef3b04dd42f1b61095385d43b597982 --- /dev/null +++ b/studies/Synechocystis/protocols/Culture conditions.md @@ -0,0 +1,4 @@ +## Culture conditions +The *Synechocystis* strains were inoculated in 30 ml BG11 liquid cultures containing 20 µg/ml spectinomycin and for overexpression strains 10 µg/ml chloramphenicol in 100 ml Erlenmeyer flasks from agar plates. The cultures were diluted twice to an OD750 of 0.2 to equalize their cell densities and growth phases. Two days before the start of the experiment, cultures were again diluted to an OD750 of 0.2 after which they were transferred to 6-well plates with 5 ml per well. L-Rhamnose was then added to the cultures and they were grown for 72 h at 30°C with 150 rpm shaking, 0.5% CO2 and 80 µE m-2 s-1 of continuous light. After 72 h, cell samples were taken and stored for further processing. + +For measurements of squalene production over time, 30 ml of BG11 were inoculated from a pre-culture to OD750 = 0.4, supplemented with 5 mM of rhamnose and incubated over 14 days. Samples were taken daily for the first four days, every second day for the following six days and after 14 days. The lost culture volume from sampling was replaced with fresh BG11 containing appropriate antibiotics and 5 mM of rhamnose. \ No newline at end of file diff --git a/studies/Synechocystis/protocols/Plasmid and strain construction.md b/studies/Synechocystis/protocols/Plasmid and strain construction.md new file mode 100644 index 0000000000000000000000000000000000000000..8788c41bc677f20b04ae76a846cc33ea1f47a34c --- /dev/null +++ b/studies/Synechocystis/protocols/Plasmid and strain construction.md @@ -0,0 +1,8 @@ +## Plasmid and strain construction +A detailed list of all relevant genetic modules and information regarding their origin, is provided in the Supporting Information (Table S1 (SI)). + +To investigate the computationally identified genes’ effect on squalene production, the pEERM4 plasmid was used to integrate each gene into the neutral site 2 (NS2) under control of the rhamnose promoter Prha (Englund et al., 2015; Behle et al., 2020). The plasmid pEERM4 Cm was a gift from Pia Lindberg (Addgene plasmid # 64026; http://n2t.net/addgene:64026; RRID : Addgene_64026) (Englund et al., 2015). This plasmid was used to clone each gene of interest under the control of Prha. It contains 500 bp DNA homologous to the upstream and downstream region of NS2, between which a chloramphenicol resistance and the gene of interest are located, flanked by the rhamnose promoter and the T7 terminator. Each gene of interest was cloned into the plasmid using the restriction enzymes NheI and PstI, with the NheI cutting site located after the start codon. The genes of interest were amplified from the Synechocystis genome, using Q5-Polymerase (NEB # M0491) according to manufacturer’s instructions with oligonucleotides shown in Table S2 (SI). In two cases, an NheI restriction site was removed from the native gene sequence without changing the amino acid sequence (gap2, sqs). The sqs gene is annotated as starting with GTG as a start codon in the published Kazusa genome and this codon was changed to ATG for the purposes of this study. + +To enable induction of the Prha promoter, the rhamnose activator rhaS was constitutively expressed by the J23119 promoter from the replicative plasmid pSHDY (AddGene Plasmid #137661, (Behle et al., 2020)), which was transferred to *Synechcoystis* via triparental mating (Behle et al., 2020). This plasmid was constructed using the restriction sites of the BioBrick and NeoBrick standards and carries a spectinomycin resistance. + +*Synechocystis* was transformed with the pEERM4 plasmids (Table S1 (SI)) using a protocol based on its natural competence (dx.doi.org/10.17504/protocols.io.mdrc256). Successful integration of the plasmid into the genome through heterologous recombination into the neutral site 2 (NS2) (Satoh et al., 2001) was verified by colony PCR (Figure S1 (SI)). The plasmid pSHDY carrying the rhamnose activator rhaS was then transferred to *Synechocystis* using triparental mating (dx.doi.org/10.17504/protocols.io.psndnde). \ No newline at end of file diff --git a/studies/Synechocystis/resources/.gitkeep b/studies/Synechocystis/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391