update2

studies/Plasmid and strain construction/protocols/Plasmid construction.md

+## 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.
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studies/Plasmid and strain construction/protocols/Promoter induction.md

+## 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.
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studies/Plasmid and strain construction/protocols/Strain construction.md

+## 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).
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studies/Synechocystis/protocols/Culture conditions.md

 ## 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.
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