Generation of GoldenBraid plasmids adapted for yeast genome integration
To generate new integration constructs compatible with the GoldenBraid2.0 standard, the backbone of GoldenBraid (GB) vectors pDGB2α1 and pDGB2Ω1 were amplified using primers 1826/1827 and 1827/1828, respectively, and new synthetic GoldenBraid cloning cassettes adapted for S. cerevisiae genome integration were ligated within. These S. cerevisiae-specific synthetic cassettes were synthetized by Genscript and consisted of recombination arms of homology for integration at the YPRCΔ15 or YORWΔ22 solo long terminal repeat (LTR) loci described in . Integration at these loci conferred a high level of expression of a reporter gene using two different promoters (TEF1p and ACT1p). These integrative arms of homology flanked the regular GB module (BsmBI-BsaI-lacZ-BsaI-BsmBI for α1 or BsaI-BsmBI-lacZ-BsmBI-BsaI for Ω1 vectors ) and the entire cassette was flanked by I-SceI restriction sites. This resulted in the construction of 4 plasmids, YPRCΔ15α1, YPRCΔ15Ω1, YORWΔ22α1 and YORWΔ22Ω1. Subsequently, the α1 vectors were mutagenized to become α2 and the Ω1 vectors were mutagenized to become Ω2, generating 8 total plasmids (Genscript).
Molecular biology enzymes
Enzymes used for molecular biology were the following: restriction enzymes BsmBI/Esp3I (Fermentas), BsaI and BtgZI (NEB), T4 DNA ligase (Promega), Phusion DNA Polymerase (Agilent) and KAPA2G (KAPA Biosystems).
Domestication of parts
GB parts were amplified by PCR from plasmid or genomic DNA templates with primers designed by using the Domesticator tool of www.GBcloning.org as described in Additional file 1: Table S1 and Additional file 2: Table S2. Domestication of GB parts is the process by which the internal restriction sites BsmBI/Esp3I, BsaI and BtgZI are removed and appropriate 4 nucleotide flanking overhangs are added to provide specificity to the part type. The sequence of each part is listed in Additional file 3: Figure S1. The DNA sequence encoding the 8xHis tag and the codon-optimized versions of the nif genes were synthetized by Genscript or Proteogenix, as described in Additional file 1: Table S1. The protocol used for the domestication reactions consists of adding 40 ng of each DNA patch, 75 ng of pUPD plasmid, 10 units of BsmBI, 3 units of T4 DNA ligase and 1 μL of 10× ligase buffer in a final volume of 10 μL. Reactions were carried out in a thermocycler, 25 cycles of 37 °C 2 min, 16 °C 5 min .
Cloning into destination plasmids
After domestication of all GB parts in the universal domesticator plasmid, pUPD, the desired transcriptional units (TUs) were generated in the α1 destination plasmids. The kanMX (G418R) or the hphMX (HygroR) resistance selection markers were digested from pUPD and subcloned into the α2 plasmids. α1-TUs and α2-selection markers were then combined into Ω1 plasmids. The protocol for the TU assembly reaction in α destination plasmids consists on adding 75 ng of each part, 75 ng of the α destination plasmid, 10 units of BsaI, 3 units of T4 DNA ligase and 1 μL of 10× ligase buffer in a final volume of 10 μL. Reactions were carried out in a thermocycler, 25 cycles of 37 °C 2 min, 16 °C 5 min . The protocol for the piling up of the TUs cloned in the two α plasmids into an Ω plasmid consists on adding 75 ng of each α plasmid, 75 ng of the Ω plasmid, 10 units of BsmBI, 3 units of T4 DNA ligase and 1 μL of 10× ligase buffer in a final volume of 10 μL. Reactions were carried out in a thermocycler, 25 cycles of 37 °C 2 min, 16 °C 5 min .
Verification of the final constructs was performed by Sanger sequencing using a promoter forward primer, a terminator reverse primer and an internal primer when needed for longer length parts.
Strains and growth media
The S. cerevisiae strains W303–1A (ura3–1; trp1–1; leu2–3112; his3–11; ade2–1; can1–100) (ATCC 208352) and CEN.PK2–1D (ura3–52; trp1–289; leu2–3112; his3Δ 1; MAL2-8C; SUC2) were used as indicated in the text and figures. Cells were grown in YPAU media (1% yeast extract (Conda), 2% bactopeptone (Pronadisa), 0.2 mg/ml adenine sulfate (Formedium) and 0.27 mg/ml uracil (Amresco)) with 2% glucose (Sigma), 2% galactose (Formedium) or 3% glycerol (GPR Rectapur VWR) as described in each case.
S. cerevisiae Transformation
The final plasmids containing the GB-assembled TU constructs were digested with I-SceI (NEB) to generate a linear DNA fragment that was used for S. cerevisiae transformation and genome integration, as described in reference .
G418 (200 μg/ml, Santa Cruz Biotechnology) and Hygromycin B (200μg/ml, Formedium) were used for selection. Transformants were purified by restreaking on YPAUD plates containing 2% glucose and the appropriate antibiotic, and only colonies growing with normal morphology were selected for further experimentation.
Verification of genomic integration was performed for each strain by extracting genomic DNA using the Bust n’ Grab Genomic DNA Isolation Protocol  and by carrying out PCR reactions for the YPRC∆15 or YORW∆22 integration loci using primers specific to sequences flanking the integration arms of homology together with internal primers specific to each construct. To confirm integration at the YPRC∆15 locus, we used the 5′ flanking forward primer 1833 and a reverse primer internal to the promoter of the nif transcription unit. To confirm YPRC∆15 3′ integration, we used a kanMX internal forward primer 1551 and 3′ flanking reverse primer 1836. To confirm YORW∆22 5′ integration, we used 5′ flanking forward primer 1829 and a reverse primer internal to the promoter of the nif transcription unit. To confirm YORW∆22 3′ integration, we used a kanMX or hphMX internal forward primer 1551 or 1556 and 3′ flanking reverse primer 1832. Primer sequences are listed in Additional file 2: Table S2.
Preparation of yeast extracts and immunoblotting
Cell pellets corresponding to OD600 5–10 were protein extracted using the alkali extraction protocol described in , and samples corresponding to OD600 0.5–1 were analyzed by SDS-PAGE and western blotting (WB). Monoclonal mouse anti-His primary antibody (Sigma H1029-2ML, 1:5000 dilution) and anti-mouse-HRP secondary antibody (Santa Cruz Biotechnology sc-2060, 1:10,000 to 1:20,000 dilution), or polyclonal rabbit anti-NifU (, 1:5000 dilution), polyclonal rabbit anti-NifS (, 1:200 dilution), polyclonal rabbit anti-NifM (1:4000 dilution), and anti-rabbit-HRP secondary antibody (Sigma A0545-1ML, 1:10,000 dilution) were used.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in .
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.
Preparation of mitochondria enriched extracts
The preparation of a crude mitochondrial fraction was performed following the protocol described in  with 5 g of cells collected from cultures grown in YPAU media with 2% glucose to a final OD600 of 4–6.
Western blots were carried out using polyclonal rabbit anti-NifU (, 1:5000 dilution), monoclonal mouse anti-HSP60 (Novus biologicals, NBP2–34671H, 1:2000 dilution), monoclonal rat anti-tubulin (Santa Cruz Biotechnology sc-69,971, 1:1000 dilution), anti-rabbit-HRP secondary antibody (Sigma A0545-1ML, 1:10,000 dilution), anti-mouse-HRP secondary antibody (Santa Cruz Biotechnology, sc-2060, 1:20,000 dilution) and anti-rat-HRP secondary antibody (Amersham NA 935, 1:10,000 dilution).
Glucose determination in culture media
Glucose presence in the media was estimated using a Glucocard G+ meter sensor.