Pet 41a

To overexpress FolE protein of R. monacensis in E. coli, the expression vector used was pET-41a(+) (EMD Millipore, Billerica, MA), which facilitates the expression of the protein as a fusion product tagged with N-terminal GST. The cloning procedure has been described previously (Bodnar et al., 2018 (link)). Briefly, the folE PCR fragment of R. monacensis and pET-41a(+) plasmid was digested with PshAI (New England Biolabs, Ipswich, MA). Ligation of the gel-purified rickettsial folE PCR amplicon in the plasmid was completed with T4 DNA ligase (New England Biolabs, Ipswich, MA), generating pET41a(+)−folE clone. After ligation, the reaction mix was transformed into competent E. coli strain NovaBlue (EMD Millipore, Billerica, MA). Kanamycin resistant colonies were then cultured in LB broth plus 100 mg/L kanamycin. The Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI) was used for plasmid purification. The insert of the pET41a(+)-folE clone was sequenced at Elim Biopharmaceuticals (Elim Biopharmaceuticals, Hayward, CA) with the following vector specific primers: forward primer 5’- AAGAAACCGCTGCTGCTAAA-3′ and reverse primer 5’- AGCTCCGTCGACAAGCTT-3’ (Elim Biopharmaceuticals, Hayward, CA).