From 4d2584c2eaf9f1a545b9476aa1a5fe04f4e2da87 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Sun, 29 Oct 2023 14:42:38 +0100
Subject: [PATCH] update2

---
 .gitattributes                                |   2 ++
 .../Data Sheet 1.PDF                          |   0
 .../fpls-14-1024981.pdf                       |   0
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6801 bytes
 .../protocols/.gitkeep                        |   0
 ...Absorption spectra and OD measurements.md} |   4 +---
 assays/DCW measurement/README.md              |   0
 assays/DCW measurement/dataset/.gitkeep       |   0
 assays/DCW measurement/isa.assay.xlsx         | Bin 0 -> 6788 bytes
 assays/DCW measurement/protocols/.gitkeep     |   0
 .../protocols/DCW measurement.md              |   2 ++
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6811 bytes
 .../protocols/.gitkeep                        |   0
 ...ents for the quantification of squalene.md |   0
 assays/Pigment quantification/README.md       |   0
 .../Pigment quantification/dataset/.gitkeep   |   0
 assays/Pigment quantification/isa.assay.xlsx  | Bin 0 -> 6793 bytes
 .../Pigment quantification/protocols/.gitkeep |   0
 .../protocols/Pigment quantification.md       |   0
 assays/Quantitative real-time PCR/README.md   |   0
 .../dataset/.gitkeep                          |   0
 .../Quantitative real-time PCR/isa.assay.xlsx | Bin 0 -> 6798 bytes
 .../protocols/.gitkeep                        |   0
 .../Quantitative real-time PCR (qRT-PCR).md   |   0
 .../isa.assay.xlsx                            | Bin 6797 -> 6797 bytes
 .../dataset/{Table 2.XLSX => TableS6.XLSX}    | Bin
 .../dataset/{Table 3.XLSX => TableS7.XLSX}    | Bin
 assays/metabolic modeling/isa.assay.xlsx      | Bin 6793 -> 6793 bytes
 .../protocols/{Table 1.XLSX => TableS5.XLSX}  | Bin
 isa.investigation.xlsx                        | Bin 9401 -> 10428 bytes
 .../README.md                                 |   0
 .../isa.study.xlsx                            | Bin 0 -> 7441 bytes
 .../protocols/.gitkeep                        |   0
 .../Quantification of squalene production.md  |   2 ++
 .../resources/.gitkeep                        |   0
 .../Plasmid and strain construction/README.md |   0
 .../isa.study.xlsx                            | Bin 0 -> 7403 bytes
 .../protocols/.gitkeep                        |   0
 .../protocols/Plasmid construction.md         |   4 ++++
 .../protocols/Promoter induction.md           |   2 ++
 .../protocols/Strain construction.md          |   2 ++
 .../resources/.gitkeep                        |   0
 studies/Synechocystis/isa.study.xlsx          | Bin 7458 -> 7538 bytes
 .../protocols/Culture conditions.md           |   1 -
 48 files changed, 15 insertions(+), 4 deletions(-)
 rename {publication => _publication}/Data Sheet 1.PDF (100%)
 rename {publication => _publication}/fpls-14-1024981.pdf (100%)
 create mode 100644 assays/Absorption spectra and OD measurements/README.md
 create mode 100644 assays/Absorption spectra and OD measurements/dataset/.gitkeep
 create mode 100644 assays/Absorption spectra and OD measurements/isa.assay.xlsx
 create mode 100644 assays/Absorption spectra and OD measurements/protocols/.gitkeep
 rename assays/{measurements and quantification/protocols/Biomass measurements (DCW, OD, spectra).md => Absorption spectra and OD measurements/protocols/Absorption spectra and OD measurements.md} (51%)
 create mode 100644 assays/DCW measurement/README.md
 create mode 100644 assays/DCW measurement/dataset/.gitkeep
 create mode 100644 assays/DCW measurement/isa.assay.xlsx
 create mode 100644 assays/DCW measurement/protocols/.gitkeep
 create mode 100644 assays/DCW measurement/protocols/DCW measurement.md
 create mode 100644 assays/GC-MS measurements for the quantification of squalene/README.md
 create mode 100644 assays/GC-MS measurements for the quantification of squalene/dataset/.gitkeep
 create mode 100644 assays/GC-MS measurements for the quantification of squalene/isa.assay.xlsx
 create mode 100644 assays/GC-MS measurements for the quantification of squalene/protocols/.gitkeep
 rename assays/{measurements and quantification => GC-MS measurements for the quantification of squalene}/protocols/GC-MS measurements for the quantification of squalene.md (100%)
 create mode 100644 assays/Pigment quantification/README.md
 create mode 100644 assays/Pigment quantification/dataset/.gitkeep
 create mode 100644 assays/Pigment quantification/isa.assay.xlsx
 create mode 100644 assays/Pigment quantification/protocols/.gitkeep
 rename assays/{measurements and quantification => Pigment quantification}/protocols/Pigment quantification.md (100%)
 create mode 100644 assays/Quantitative real-time PCR/README.md
 create mode 100644 assays/Quantitative real-time PCR/dataset/.gitkeep
 create mode 100644 assays/Quantitative real-time PCR/isa.assay.xlsx
 create mode 100644 assays/Quantitative real-time PCR/protocols/.gitkeep
 rename assays/{measurements and quantification => Quantitative real-time PCR}/protocols/Quantitative real-time PCR (qRT-PCR).md (100%)
 rename assays/metabolic modeling/dataset/{Table 2.XLSX => TableS6.XLSX} (100%)
 rename assays/metabolic modeling/dataset/{Table 3.XLSX => TableS7.XLSX} (100%)
 rename assays/metabolic modeling/protocols/{Table 1.XLSX => TableS5.XLSX} (100%)
 create mode 100644 studies/Measurements of squalene production/README.md
 create mode 100644 studies/Measurements of squalene production/isa.study.xlsx
 create mode 100644 studies/Measurements of squalene production/protocols/.gitkeep
 create mode 100644 studies/Measurements of squalene production/protocols/Quantification of squalene production.md
 create mode 100644 studies/Measurements of squalene production/resources/.gitkeep
 create mode 100644 studies/Plasmid and strain construction/README.md
 create mode 100644 studies/Plasmid and strain construction/isa.study.xlsx
 create mode 100644 studies/Plasmid and strain construction/protocols/.gitkeep
 create mode 100644 studies/Plasmid and strain construction/protocols/Plasmid construction.md
 create mode 100644 studies/Plasmid and strain construction/protocols/Promoter induction.md
 create mode 100644 studies/Plasmid and strain construction/protocols/Strain construction.md
 create mode 100644 studies/Plasmid and strain construction/resources/.gitkeep

diff --git a/.gitattributes b/.gitattributes
index 97e6e92..63a9a4c 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 0000000..e69de29
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 0000000..e69de29
diff --git a/assays/Absorption spectra and OD measurements/isa.assay.xlsx b/assays/Absorption spectra and OD measurements/isa.assay.xlsx
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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 0000000..e69de29
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 df136c9..222ba18 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
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diff --git a/assays/DCW measurement/protocols/.gitkeep b/assays/DCW measurement/protocols/.gitkeep
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diff --git a/assays/DCW measurement/protocols/DCW measurement.md b/assays/DCW measurement/protocols/DCW measurement.md
new file mode 100644
index 0000000..6bf234d
--- /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
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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
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diff --git a/assays/measurements and quantification/protocols/GC-MS measurements for the quantification of squalene.md b/assays/GC-MS measurements for the quantification of squalene/protocols/GC-MS measurements for the quantification of squalene.md
similarity index 100%
rename from assays/measurements and quantification/protocols/GC-MS measurements for the quantification of squalene.md
rename to assays/GC-MS measurements for the quantification of squalene/protocols/GC-MS measurements for the quantification of squalene.md
diff --git a/assays/Pigment quantification/README.md b/assays/Pigment quantification/README.md
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diff --git a/assays/Pigment quantification/dataset/.gitkeep b/assays/Pigment quantification/dataset/.gitkeep
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diff --git a/studies/Measurements of squalene production/protocols/.gitkeep b/studies/Measurements of squalene production/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
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 0000000..9d64d14
--- /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
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diff --git a/studies/Plasmid and strain construction/README.md b/studies/Plasmid and strain construction/README.md
new file mode 100644
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diff --git a/studies/Plasmid and strain construction/protocols/.gitkeep b/studies/Plasmid and strain construction/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
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 0000000..2b18216
--- /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 0000000..1ee6926
--- /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 0000000..6b62ced
--- /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 0000000..e69de29
diff --git a/studies/Synechocystis/isa.study.xlsx b/studies/Synechocystis/isa.study.xlsx
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diff --git a/studies/Synechocystis/protocols/Culture conditions.md b/studies/Synechocystis/protocols/Culture conditions.md
index 257fbb9..bc22e9a 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
-- 
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