Pb no3 2

Trypsin-EDTA, Dulbecco’s modified Eagle’s medium (DMEM/F-12 (1:1)), fetal bovine serum (FBS, 10%), penicillin-streptomycin (PEN-STREP), zinc nitrate hexahydrate, cadmium nitrate tetrahydrate and Pb(NO₃)₂ were purchased from Sigma. Commercial Pb, Cd and Zn standards (1 g l⁻¹) were prepared from Merck. Graphene quantum dots (blue luminescent) were purchased from Sigma-Aldrich. The other chemicals which were needed for doing this project were available in archive of our laboratory which had been purchased from Sigma or Merck. Doubly distilled water was used wherever water was needed. A phosphate buffer solution (PBS, 0.01 M) was prepared from Na₂HPO₄ and its pH was adjusted at 7.4 by the use of H₃PO₄ and NaOH.

DNA polymerase, restriction endonucleases, T4 DNA ligase, and all primers were purchased from/synthesized by Takara corporation (Dalian, China). Sucrose, ZnSO₄⋅7H₂O, MnSO₄⋅H₂O, and AgNO₃ were obtained from Xilong Chemical Industry Corporation (Shanghai, China). Ni₃SO₄⋅6H₂O, MgCl₂⋅6H₂O, and CuSO₄⋅5H₂O were purchased from Xinbao Fine Chemical Factory (Shanghai, China). HgCl₂ was purchased from Beisite Chemical Reagent Corporation (Chengdu, China). 3-(N-Morpholino)propanesulfonic acid (MOPS), Pb(NO₃)₂, FeC₆H₅O₇, (NH₄)₂Fe(SO₄)₂, and CoCl₂⋅6H₂O were purchased from Sigma-Aldrich (Shanghai) Trading Corporation. The standard copper solution (1,000 μg/ml) was obtained from the National Standard Substances Center (Beijing, China). Other chemicals used were of analytical grade. All the medium and buffer solutions were prepared using deionized distilled water (Millipore, United States). All tube cultures of bacteria were soaked in potassium dichromate solution for at least 24 h to remove residual heavy metals.

Ludwigia stolonifera (Guill.&Perr.) was obtained from the mainstream of the River Nile bank, Sohag, Egypt and they transferred to the laboratory. Because of the lack of anthropogenic activity and the low levels of metals detected, preliminary measurements (Pb concentration < 25 mg/kg in bottom sediment) in this location were used as a contamination control site [49 (link)]. To eliminate clinging particles, the plants were rinsed numerous times with tap and distilled water. Plants with identical biomass and height were chosen and stored separately in 20 L aquariums with half strength Hoagland’s solution with a pH of 7 [50 ] for 15 days before acclimatization. Then, the acclimatized plants were transferred and maintained in aquariums with 5% Hoagland’s solution including varied concentrations of working Pb standard solutions 0, 10, 25, 50, and 100 mg/L. They were then exposed to Pb concentrations over a period of time (0, 1, 3, 7, and 10 days). Pb of analytical grade was provided; Pb(NO₃)₂ (Sigma, St. Louis, MO, USA) was used as the source of Pb. The trials were carried out in a controlled temperature (24 ± 2 °C by using XH-M452 Thermostat Temperature Humidity Control Sw) and with light (1950 Lux).

E. coli and B. subtilis strains, as well as the plasmids used and generated in this study, are described in Table 1. Lysogeny broth (LB; Lennox formulation) was used for the bacterial liquid or solid cultures. When required, chloramphenicol (Cm; 5 μg/mL), ampicillin (Amp; 100 μg/mL), and/or bacteriological agar (15 μg/mL) were added to the medium. All cultures were incubated at 37°C with agitation (250 rpm) for liquid cultures. The cell optical density (OD) of liquid cultures was monitored with the spectrophotometer Thermo Scientific Genesis set at 600 nm.
We formulated 10-mM stock solutions of As(V), As(III), DMA(V), and Cr(VI) formulated with Na₂HAsO₄·7H₂O, NaAsO_2, (CH₃)₂AsO₂Na·3H₂O, and K₂Cr₂O_7, respectively. For Cd(II), Pb (II), and Zn(II), 1,000 ppm stock standard solutions of Cd(NO₃)₂·4H₂O₂, Pb(NO₃)₂, and Zn(NO₃)₂·6H₂O from Sigma-Aldrich were used, respectively.