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]