diff --git a/.gitattributes b/.gitattributes index 97e6e92d510c5f6a4542778fa02543aaae1dd365..63a9a4ccb788a65863caf7bb48d29d386e8e66bd 100644 --- a/.gitattributes +++ b/.gitattributes @@ -1,2 +1,4 @@ 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 +_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/publication/Data Sheet 1.PDF b/_publication/Data Sheet 1.PDF similarity index 100% rename from publication/Data Sheet 1.PDF rename to _publication/Data Sheet 1.PDF diff --git a/publication/fpls-14-1024981.pdf b/_publication/fpls-14-1024981.pdf similarity index 100% rename from publication/fpls-14-1024981.pdf rename to _publication/fpls-14-1024981.pdf diff --git a/assays/Absorption spectra and OD measurements/README.md b/assays/Absorption spectra and OD measurements/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/Absorption spectra and OD measurements/dataset/.gitkeep b/assays/Absorption spectra and OD measurements/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/Absorption spectra and OD measurements/isa.assay.xlsx b/assays/Absorption spectra and OD measurements/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..daba8e9df45d7f505fc6c052cad989f2426742cd Binary files /dev/null and b/assays/Absorption spectra and OD measurements/isa.assay.xlsx differ diff --git a/assays/Absorption spectra and OD measurements/protocols/.gitkeep b/assays/Absorption spectra and OD measurements/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/Absorption spectra and OD measurements/protocols/Absorption spectra and OD measurements.md similarity index 51% rename from assays/measurements and quantification/protocols/Biomass measurements (DCW, OD, spectra).md rename to assays/Absorption spectra and OD measurements/protocols/Absorption spectra and OD measurements.md index df136c9d4ec45786f604b883d838310e64c2d4e9..222ba1862493f0552b10227729abbe93308450e4 100644 --- a/assays/measurements and quantification/protocols/Biomass measurements (DCW, OD, spectra).md +++ b/assays/Absorption spectra and OD measurements/protocols/Absorption spectra and OD measurements.md @@ -1,4 +1,2 @@ -## 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 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/DCW measurement/README.md b/assays/DCW measurement/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/DCW measurement/dataset/.gitkeep b/assays/DCW measurement/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/DCW measurement/isa.assay.xlsx b/assays/DCW measurement/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..f4907fafb3acdd7d4d624454ebf78fa9da13e5e2 Binary files /dev/null and b/assays/DCW measurement/isa.assay.xlsx differ diff --git a/assays/DCW measurement/protocols/.gitkeep b/assays/DCW measurement/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/DCW measurement/protocols/DCW measurement.md b/assays/DCW measurement/protocols/DCW measurement.md new file mode 100644 index 0000000000000000000000000000000000000000..6bf234ddf95f46eea023864f88b3fb15ffa9360a --- /dev/null +++ b/assays/DCW measurement/protocols/DCW measurement.md @@ -0,0 +1,2 @@ +## 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-MS measurements for the quantification of squalene/README.md b/assays/GC-MS measurements for the quantification of squalene/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/GC-MS measurements for the quantification of squalene/dataset/.gitkeep b/assays/GC-MS measurements for the quantification of squalene/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/GC-MS measurements for the quantification of squalene/isa.assay.xlsx b/assays/GC-MS measurements for the quantification of squalene/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..99179e733e09e1e72913569caf719043eb9e3d90 Binary files /dev/null and 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0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Measurements of squalene production/protocols/Quantification of squalene production.md b/studies/Measurements of squalene production/protocols/Quantification of squalene production.md new file mode 100644 index 0000000000000000000000000000000000000000..9d64d14dee6d3022539b43f126c56a60f1879431 --- /dev/null +++ b/studies/Measurements of squalene production/protocols/Quantification of squalene production.md @@ -0,0 +1,2 @@ +## Quantification of squalene production +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/Measurements of squalene production/resources/.gitkeep b/studies/Measurements of squalene production/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Plasmid and strain construction/README.md b/studies/Plasmid and strain construction/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Plasmid and strain construction/isa.study.xlsx b/studies/Plasmid and strain construction/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..21dd949b81129d3b87c154fb5b3015ee02a8d9f4 Binary files /dev/null and b/studies/Plasmid and strain construction/isa.study.xlsx differ diff --git a/studies/Plasmid and strain construction/protocols/.gitkeep b/studies/Plasmid and strain construction/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Plasmid and strain construction/protocols/Plasmid construction.md b/studies/Plasmid and strain construction/protocols/Plasmid construction.md new file mode 100644 index 0000000000000000000000000000000000000000..2b182164161b58dc11f8b806e7fbf6dea12139dc --- /dev/null +++ b/studies/Plasmid and strain construction/protocols/Plasmid construction.md @@ -0,0 +1,4 @@ +## Plasmid 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. \ No newline at end of file diff --git a/studies/Plasmid and strain construction/protocols/Promoter induction.md b/studies/Plasmid and strain construction/protocols/Promoter induction.md new file mode 100644 index 0000000000000000000000000000000000000000..1ee69266f6e4821e03338b539fc73baa1d963901 --- /dev/null +++ b/studies/Plasmid and strain construction/protocols/Promoter induction.md @@ -0,0 +1,2 @@ +## Promoter induction +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. \ No newline at end of file diff --git a/studies/Plasmid and strain construction/protocols/Strain construction.md b/studies/Plasmid and strain construction/protocols/Strain construction.md new file mode 100644 index 0000000000000000000000000000000000000000..6b62ced692cde591c0ffb4f0e549be6986fbf44f --- /dev/null +++ b/studies/Plasmid and strain construction/protocols/Strain construction.md @@ -0,0 +1,2 @@ +## Strain construction +*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/Plasmid and strain construction/resources/.gitkeep b/studies/Plasmid and strain construction/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/Synechocystis/isa.study.xlsx b/studies/Synechocystis/isa.study.xlsx index 3aeed844765339c37fd19617c9ab8f70f3ca6679..9ecf9e8fd24761f4901a44fc7b89cbbda92b0738 100644 Binary files a/studies/Synechocystis/isa.study.xlsx and b/studies/Synechocystis/isa.study.xlsx differ diff --git a/studies/Synechocystis/protocols/Culture conditions.md b/studies/Synechocystis/protocols/Culture conditions.md index 257fbb925ef3b04dd42f1b61095385d43b597982..bc22e9aa58a27afc0f7a8398e121aff56e03e863 100644 --- a/studies/Synechocystis/protocols/Culture conditions.md +++ b/studies/Synechocystis/protocols/Culture conditions.md @@ -1,4 +1,3 @@ ## 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