4.2 Plasmid transformation
For plasmid amplification, 1 μL plasmid (approximately 100 ng
μL-1) was transformed to 50 μL E. coli DH5ɑ
competent strain (TIANGEN). It was then incubated on ice for 25 minutes
followed by 90 seconds heat shock at 42℃. Subsequently, it was mixed
with 450 μL LB medium and incubated for an hour using a rotary shaker
(ZQZY-BG, Shanghai Zhichu). For protein expression, E. coliBL21(DE3) strain was employed (TIANGEN and TSINGKE). 100 μL Competent
cell culture was used for co-transformation of two plasmids, the
cultures were added with 900 μL LB medium to recover the cells. For
single plasmid transformation, the protocol was the same as for E.
coli DH5ɑ amplification. For plasmid construction using HiFi assembly
(NEB), 5 μL product were transformed to 100 μL bacterial culture,
followed the same protocol as that for amplification.