2.4 Real-time quantitative PCR (qPCR) and calculation of
relative translation efficiency
To compare the EGFP transcriptional level of different plasmids,E. coli strains were in LB medium as described above. 1 mL of
cultures was collected by centrifugation and washed twice with
ice-cooled PBS. Total RNA extraction (CWBIO, China), reverse
transcription (Vazyme, China), and qPCR (Vazyme, China) were then
performed according to the manufacturer’s instructions. qPCR was carried
out using StepOnePlus (Applied Biosystem, USA) system and the condition
was set as follows: 95 °C for 30 s and 40 cycles at 95 °C for 15 s, 62
°C for 30 s, and 72 °C for 20 s. Each sample contains three biological
repeats and three duplicated wells, the relative EGFP transcription
level was analyzed by the 2−ΔΔCtmethod.[19] The 16S rRNA was used as the
endogenous control and The EGFP transcription level of pET28-EGFP was
defined as 1. The relative EGFP translation efficiency is calculated by
dividing the EGFP fluorescence intensity by mRNA abundance as previously
reported.[24,25]