Induction of T7 Promoter at Higher Temperatures May Be Counterproductive

摘要

Flow cytometry and Western blot analysis showing the effect of T7 promoter induction on protein expression at various temperatures. a Representative histograms of flow cytometry experiments (n = 6) showing the percentage of emGFP positive cell of samples that are either uninduced or induced with IPTG at difference temperatures as indicated. The histogram of untransformed cells is shown as control. The flow cytometry data was collected by exciting the cells at 488 nm and measuring fluorescence at measured at 530 ± 10 nm of 10,000 events. The histogram clearly shows that percentage of emGFP positive cells have significantly reduced when induced at higher temperatures. b Dot plots corresponding to the histogram are shown. c The bar graph is the average data of flow cytometry experiments (n = 6) comparing the percentage of emGFP positive cells of samples that were uninduced (UI) with induced (IN) samples at various temperatures as mentioned in the graph. To compare the reduction of emGFP positive cells in induced samples graph was plotted by normalizing the percentage of fluorescence of induced samples to percentage of emGFP positive cells of uninduced samples. d The western blot analysis to assess the effect of induction on protein expression at various temperatures was performed using Xpress epitope. The upper panel is the western blot of weight normalized whole cell lysate of samples with and without induction at various temperatures as mentioned in the figure. The lower panel is the western blot of soluble fraction showing the amount of protein present in samples as mentioned in the figure