2.1 Materials and reagents
CdCl2, CoCl2·6H2O, HgSO4, CuCl, and meso-2,3-Dimercaptosuccinic acid (DMSA) were all purchased from Macklin (Shanghai, China). KOD FX was purchased from Toyobo (Shanghai, China), and homologous recombinase was purchased from Clone Smarter (USA). T4 PNK ligase was purchased from Takara (Japan). In this study, other unspecified reagents were of analytical grade. CadR (NCBI: AF333961) was synthesized by GENEWIZ (Suzhou, China). pRSETB was from Invitrogen.
2.2 Construction of cadmium sensor
We ligated CadR to pRSETB by homologous recombination, and then performed reverse PCR between the C112 to C119 amino acids of pRSETB-CadR (Table S1), and using the same method amplified cpYFP with the homology arms at both ends of the insertion point of CadR the linkers SAG and GTG were added (Table S2). Finally, the homologous recombinase was used for ligation for 15 mins at 50°C, and then transferred to Escherichia coli Top10 competent strain for amplification, and then extracted and sequenced. The plasmids identified by sequencing were introduced into E. coli BL21(DE3) competent cells, incubated overnight at 37°C, a single colony was picked to inoculate 100 ml of LB medium, and 0.1 mg/ml of ampicillin was added. Following culture for 8–12 hours at 37°C and 220 rpm to an OD600 of 0.4–0.6, 1 mM Isopropyl-beta-D-thiogalactopyranoside was added and incubated afor 20–24 hours t 18°C. The fermented strains were collected into HEPES buffer (100 mM HEPES, 100 mM NaCl) via ultrasonication, and the cell lysate supernatant was diluted with HEPES buffer, 0.5 µM, and 5 µM CdCl2 were added, and the fluorescence intensity was immediately detected at F485/528 and F420/528.
To further optimize the cadmium sensor, we firstly knocked out the N-terminal methionine of cpYFP, and then used traditional truncation methods to shorten the amino acid linker between cpYFP and CadR to improve the response of CadR16 (Table S3 and S4). First, we used reverse PCR to remove the amino acid linker at the N-terminus of cpYFP in CadR16. The C-terminal linker was then reduced by the same method. In our nomenclature, N and C are the abbreviations for N-terminal and C-terminal, respectively. Therefore, N1C1 means that one amino acid at the N-terminal and C-terminal of the cpYFP linker have been removed from CadR16. All these truncated mutants were screened as described above. In addition, we changed the key cysteines C77, C112, and C119 of N0C0 to serine by site-directed mutagenesis (Table S5).
2.3 Protein expression and purification
We dissolved E. coli containing N0C0, N1C1, and C112S proteins in HEPES buffer and sonicated them on ice. These fusion proteins all had 6× His tags when they were constructed, and were purified by nickel-column affinity chromatography (Cytiva) on the AKTA pure system. The eluents were incubated with 10 mM EDTA at 4 ℃ to remove possible metal ions, such as nickel. Then, indicator proteins were concentrated using the Amicon Ultra centrifugal filter device (Millipore). A 5 ml desalting column (Cytiva) was then used to remove the imidazole and chelating agent contained in it, and was equilibrated with HEPES buffer (100 mM, 100 mM NaCl, pH 7.4) and then HEPES buffer (10 mM, 100 mM NaCl, pH 7.4). Protein concentrations were determined by the Bradford method using Coomassie Protein Assay Reagent with bovine serum albumin as the standard.
2.4 In vitro characteristics of the cadmium sensors
For spectrum measurement, the purified 0.2 µM N0C0 and N1C1 were added with or without 0.5/5 µM CdCl2, and a microplate reader (TECAN infinite M200) was then used to measure the absorption spectrum at room temperature. The extinction coefficient was calculated with the Beer-Lambert equation according to the absorption spectrum. The fluorescence spectrum was detected with the emission wavelength fixed at 530 nm and the excitation wavelength at 400–550 nm; the excitation wavelength was fixed at 485 nm, the emission wavelength was 485–600 nm, and the scanning interval was 1 nm. To determine the quantum yield of purified N0C0 and N1C1, excitation was performed at the ultraviolet absorption peak, the emission spectrum was measured with a fluorescence spectrophotometer (Shimadzu RF-6000), and the integrated fluorescence value was calculated. EGFP (QY 0.60, pH 7.4) was used as a control to calculate the quantum yield of N0C0. Similarly, the quantum yield of N1C1 was measured and calculated using the Brightness and Fluorescence Changes.
For all microplate experiments, the recombinant protein was diluted to a final concentration of 0.1–0.2 µM, and fluorescence was detected using the same settings as those for screening. In the Cd2+ titration experiment, 100 µL of different concentrations of Cd2+ and 100 µL of protein were mixed in a 96-well flat-bottomed plate, and the fluorescence change was immediately measured. All fluorescence intensities were normalized to a signal of 1 in the absence of cadmium at pH 7.4 and data were fitted to the Hill1 equation.
To determine the specificity of N0C0 and N1C1 against other ions, 100 µL of buffer containing different ionic components and 100 µL of purified protein were used for the reaction with or without 0.5/5 µM CdCl2. The ion concentrations are listed below: 300 mM Na+ or K+, whereas other ions were 100 µM (Fe3+, Mg2+, Ca2+, Ni2+, Mn2+, Fe2+, Li+, Cs+, Ba2+, Co2+) or 0.5 µM (Ag+, Zn2+, Cu2+, Pb2+, Hg+, Cu+). To determine the sensitivity of N0C0 and N1C1 to temperature, a 25–40°C temperature program in a microplate reader was performed and the fluorescence change was detected every 20 seconds. To determine the dependence of N0C0 and N1C1 on the pH value, HEPES buffer with a pH value ranging from 6.8 to 8.4 was prepared at 0.2 pH unit intervals.
At the same time, in order to study the in vitro response kinetics of N0C0 and N1C1, 0.5/5 µM Cd2+ and 2 mM DMSA were sequentially added, and the fluorescence changes were monitored every 20 seconds.
2.5 Measuring inorganic cadmium in E. coli
E. coli cells after fermentation were washed and dissolved in HEPES buffer, the bacterial solution was diluted to an OD600 of 1, the microplate reader was set to 37°C and incubated for 5 min, and 0.5/5 µM Cd2+ and 2 mM DMSA were sequentially added. The fluorescence changes were detected every 60 seconds.
2.6 Data analysis
Unless otherwise specified in this study, data processing was normalized, and the ratio of excitation at 485 nm, excitation at 420 nm, and emission at 528 nm (R485/420) are presented. The data are presented as a representative example of a single experiment repeated three or more times. The data obtained are expressed as mean ± SD or mean ± SEM.