Escherichia coli BL

5.4. cDNA cloning and heterologous expression in E. coli Plants of R. lindenbergiana were harvested for RNA extraction using the QIAGEN RNA isolation Kit. RNA was then converted to single-strand cDNA by GE First-strand cDNA synthesis Kit. The RlMTPSL genes were amplified by RT-PCR using the primers listed in Supplemental Table S1. The amplification products were cloned into the protein expression vector pEXP5-CT/TOPO (Invitrogen) and were confirmed by sequencing. Protein expression constructs were transferred into E. coli BL 21-Codon Plus (DE3) for protein expression and the bacteria were cultured in LB medium at 37 ◦ until OD 600 reached 0.4–0.6. The recombinant proteins were expressed for 16 h at 18 ◦ C by adding 0.4 mM isopropyl-b-D-1-thiogalactopyranoside (IPTG). The cells were collected by centrifugation at 4000 g for 10 min. After that, they were disrupted by a 8 × 10 s treatment with a sonicator (Misonix Microson ultrasonic cell disruptor, USA) in chilled extraction buffer [50 mM Tris⋅HCl, pH 7.5, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10% (vol/vol) glycerol]. Cell fragments were removed by centrifugation at 14,000 g for 30 min and the supernatant was desalted into assay buffer.Published as part of Fan, Honghong, Wei, Guo, Chen, Xinlu, Guo, Hong, Crandall-Stotler, Barbara, Kollner, Tobias G. & Chen, Feng, 2021, Sesquiterpene biosynthesis in a leafy liverwort Radula lindenbergiana Gottsche ex C. Hartm, pp. 1-8 in Phytochemistry (112847) (112847) 190 on page 7, DOI: 10.1016/j.phytochem.2021.112847, http://zenodo.org/record/827303