Dna sequencing

MRE11 sequences were inserted into the vector pLenti-puro-CMV (P100022; Vigene, Rockville, MD, USA). A QuickChange II XL Site-Directed Mutagenesis Kit (#200521; Agilent, Santa Clara, CA, USA) and XL10-Gold ultracompetent cells were used for all site-directed mutageneses according to the manufacturer’s instructions. DNA sequencing (Source Bioscience, Nottingham, UK) was used to confirm the mutated nucleotides and the deleted exon 16 sequences (Supplementary Figure 1).

All C. reinhardtii cell lines used in this research were made from the parental line TN72 [40 (link)], which is a cw15 (i.e. cell wall deficient) mutant with an aadA spectinomycin resistance cassette replacing part of psbH in the chloroplast genome. TN72 is available from the Chlamydomonas Resource Center (http://www.chlamycollection.org) as strain CC-5168. Transformation of TN72 was performed using the glass bead vortexing method [40 (link)] and a plasmid containing an intact copy of psbH in addition to the genes of interest, resulting in markerless phototrophic transformants whose homoplasmicity was confirmed by PCR as described previously [35 (link)]. DNA sequencing (Source BioScience, Nottingham, UK) was used to confirm that the introduced genetic elements were intact. C. reinhardtii was grown in Tris-acetate phosphate (TAP) medium [60 ], with 2% agar (Fisher Bioreagents, New Hampshire, USA) added for solid plates. For selection of phototrophic transformants, high-salt minimal (HSM) medium with 2% agar was used [60 ]. Liquid cultures were grown in glass flasks or in clear plastic 12 well tissue culture plates (VWR, Pennsylvania, USA). For some experiments (see Additional file 1: Table S2), two parallel Algem photobioreactors were used (Algenuity, Stewartby, UK).

The changes to the nisA genes within the corresponding pDF05 (pCI372-nisA) [24 (link)] derivatives were established through DNA sequencing (SourceBioscience, Waterford, Ireland) using the primers pCI372For 5′-CGGGAAGCTAGAGTAAGTAG-3’ and pCI372Rev 5′-ACCTCTCGGTTATGAGTTAG-3′. Sequence alignments with the nisA gene were carried out with Lasergene Megalign 7.00 (DNAStar) to determine the nature of the codon changes.