Phusion high fidelity polymerase

The 18S rRNA sequence for strain C596 was amplified using a set of universal eukaryotic primers [41 (link)] (S1 Table). The thermal cycling protocol was designed following the manufacturer’s instructions for the high-fidelity Phusion polymerase (New England Biolabs, Ipswich, MA, USA). The resulting amplicon was sequenced using the Sanger method at the Cornell University Core Genomics Facility. The phylogenetic relationship of the resulting 1800 bp 18S rRNA sequence for Chlorella strain C596 is determined through a distance tree created in MEGA 6 (http://www.megasoftware.net/) using Muscle. The sequences that strain C596 were compared against are included in S1 File.

Plasmids and primers used in this study are listed in Tables 2, 3, respectively. Primers were synthesized by Sigma-Aldrich, United States. PCR amplifications were carried out using high fidelity Phusion polymerase (New England Biolabs, United States) or DreamTaq DNA polymerase (ThermoFischer Scientific, United States). Plasmid isolation carried out using Monarch Plasmid Miniprep Kit (New England Biolabs, United States) and genomic DNA preparations were carried out using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, United States) following the manufactures instructions. Restriction enzymes were supplied by New England Biolabs and were used according to the manufacturers’ instructions. Electroporation of C. beijerinckii was performed as described previously (Oultram et al., 1988 (link)).

Mutations in the putative CRE/AP-1-like motif and GATA-binding site were generated in both pGL3/600 and pGL2-TATA-150 vectors by reverse PCR using High fidelity Phusion polymerase (New England Biolabs, Pickering, Canada) following manufacturer’s instructions. Oligonucleotides used to generate these mutations by PCR amplification are indicated in Table 1 as M1, M5 and M6.

Yeast genomic DNA was purified by lysing cells by glass bead disruption, followed by phenol/chloroform extraction and ethanol precipitation (Hoffman and Winston, 1987 (link)). Wolbachia DNA was purified by homogenizing 10 whole infected insects in lysis buffer and recovering DNA with organic extraction following referenced protocols (Beckmann et al., 2017 (link); Beckmann and Fallon, 2012 (link)). Drosophila melanogaster and D. simulans lines infected with wMel, wRi, and wHa were used as PCR template sources. In some cases, genes from wNo and wMel were subcloned from synthesized constructs (Genscript). Genomic wStr DNA was a gift from Ann Fallon and was derived from infected cell cultures. PCR amplicons were produced with primers listed in Supplementary file 1i. High fidelity Phusion polymerase (New England Biolabs) was used to amplify DNA, which was then restriction enzyme digested, gel-purified and ligated into various plasmid vectors (Supplementary file 1j). Plasmids were sequenced and confirmed at the Yale Keck Foundation DNA sequencing facility. Point mutations were introduced by QuikChange site-directed mutagenesis (Stratagene). Other modifications such as truncations or tag additions/swaps were created by site-directed ligase-independent mutagenesis (SLIM) (Chiu et al., 2004 (link)).

The coding sequence of feoB was PCR-amplified from both Iowa and RML LVS using the high fidelity Phusion polymerase (New England Biolabs). Sanger sequencing was performed to confirm that the SNP observed in the RML LVS background was present. The feoA coding sequence was cloned into the KpnI/SalI sites of a derivative of pTrc99a, immediately downstream of the groE promoter. The entire P_groE-feoA fragment was removed by digestion with BamHI and SalI, and ligated into the same sites in pBB103. Both this plasmid and the pWKS30 containing feoB were transformed into the E. coli H1771 strain (MC4100 aroB feoB7 fhuF::plac Mu). Control strains were also generated that carried only one of the plasmids. Miller assays were performed to measure FeoB Fe²⁺ import activity via the readout of Fur repression of fhuF::placZ. All strains were grown in LB supplemented with 50 μg/mL of spectinomycin and 100 μg/mL of ampicillin, as necessary.