Escherichia coli, BL, BL

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, Phytochemistry (112847) 190, pp. 1-8 : 7

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.