Cytation 3 imaging reader system

The pH_in of cells were assayed using a pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein succinimidyl ester (BCECF-AM) (Beyotime Institute of Biotechnology, China) [11 (link)]. Firstly, cells during different periods were harvested by centrifugation at 14,972g for 10 min. Then the cell pellets were resuspended in PBS buffer (50 mM K₂HPO₄, 50 mM KH₂PO₄, pH 7.0), washed twice and diluted to an OD₆₀₀ of 3.0. Secondly, 400 µL of the above bacterial suspension and 4 µL valinomycin were added to brown tubes and incubated at 30 °C for 30 min. Thirdly, 1 µL of BCECF-AM was added into the brown tubes and incubated at 30 °C for 20 min; then 200 µL of the reaction solution was taken out and centrifuged at 14,972g for 5 min. Lastly, 150 µL of the reaction solution and the supernatant were taken out to measure the fluorescence intensity. Measurements of the fluorescence intensity were performed using a Cytation 3 imaging reader system (BioTek, Winooski, VT, USA). The excitation wavelengths were 490 and 440 nm. The emission wavelength was 525 nm. The relative fluorescence intensity (RFI) was calculated as follows: RFI = [(I₄₉₀)_total − (I₄₉₀)_supernate]/[(I₄₄₀)_total − (I₄₄₀)_supernate]. Based on the values of lg (RFI), the intracellular pH was calculated from the standard curve. Measurements were performed with three biological replicates.

The recombinant strains for GFP production or the fusion libraries cultured in the 96-wells or shake flasks under the corresponding culture conditions were harvested and washed twice by phosphate buffer solution (PBS, 50 mM, pH 7.5). Whole cell fluorescence and cell density (OD₆₀₀) were measured on a Cytation 3 imaging reader system (BioTek, Winooski, VT, USA). The corresponding wild-type strain E. coli BL21 (DE3) was used as the negative control, and its fluorescence intensity was subtracted as the background. The emission and excitation wavelength of GFP were 520 and 488 nm, respectively.