Cultivation Of Strains
E. coli Jm109 was used as the control strain in our study. Cells were grown in Luria Bertani (LB) medium containing beef extract (10 g/L), peptone extract (10 g/L) and NaCl (3 g/L) at 37°C with 150 rpm shaking. Recombinant cells (MT2A and MT3) were seeded in LB medium containing ampicillin (Merc), unlike the control Jm109 cell.
Preparation Of Cr(Vi) Solution
Chemicals used in the preparation of solutions for stock metals were obtained from Merc. In this study, Cr(VI) stock solution was prepared by dissolving potassium dichromate (K2Cr2O7) metal salt in distilled water. Then, the Cr(VI) solution was sterilized in autoclaves and stored at + 4 0C.
Gene Synthesis, Cloning And Bacterial Transformation
The human MTs (MT2A and MT3) gene sequences were provided from Twist Bioscience. MT2A and MT3 gene fragments and the pET21 plasmid were double digested with EcoRI and NotI restriction enzymes (Thermo Scientific). Then these genes were individually ligated to the pET21 plasmid. Host Jm109 strain (ATCC 53323) was made competent according to the chemical CaCl2 protocol (Merc). Recombinant pET21 plasmids (Sigma-Aldrich) were transformed into competent Jm109 cells. Then, recombinant (MT2A and MT3) strains were seeded on LB agar plates supplemented with ampicillin (50 µg/ml), X-gal (for blue/white cell screening) and IPTG and incubated at 37°C overnight. The plasmid was isolated using the QIAGEN Qiaprep Spin Miniprep Kit (Cat no: 27104), and the presence of the positive transformant was confirmed using PCR. To validate additional genes, sequencing libraries of isolated plasmids were prepared and sequenced using Miseq platform (Table 1).
Table 1
Sequencing libraries for the isolated plasmids
Name | Tube ID | Insert Length | Construct Length | Insert Sequence | Construct Sequence | Yield (ng) | NGS | Yield | Expression Vector |
MT3 | tSHPs0903B | 300 | 5646 | ATGGACCCT | ATGGACCCT | 612 | PASS | PASS | pET-21 |
MT2 | tSHPs0903B | 300 | 5646 | ATGGATCCC | ATGGATCCC | 523 | PASS | PASS | pET-21 |
Minimal Inhibitory Concentration (Mic) Of Cells Against Cr(Vi)
The Cr(VI) heavy metal ion resistance capacities of Jm109 and its recombinant strains (MT2A and MT3) were determined by the minimum inhibition concentration. Minimum inhibition concentration experiments were performed with a few minor changes in the method applied by Anusha and Natarajan (2020). Three independent control groups (just LB, LB and metal solution and LB and cell culture) were used in the analysis. Cells were incubated in LB medium containing different concentrations of Cr(VI) (0-1000 mg/L) at 37°C for 96 hours with shaking to determine the Cr(VI) MIC concentration. Culture samples were taken from the medium at regular time intervals (every 8 hours) and measured at 600 nm in the microplate reader (Thermo Flash 2000).
Residue of Cr in the medium and bioreduction of Cr(VI)
In the bioaccumulation experiments, the method of Shamim et al. (2014) was modified and used. The strains were incubated at 37°C and 150 rpm for 24 hours in LB broth containing Cr(VI) (0.05-25 mg/L). Samples were taken after the incubation was completed to determine the amount of Cr remaining in the medium. The samples were centrifuged and the supernatant was filtered with a syringe filter. Total Cr accumulations in the samples were measured by inductively coupled plasma mass spectrometry (ICP-MS) (PerkinElmer, USA & Nexion 350X). The Cr(VI) concentration of the supernatant was analyzed at 540 nm by the 1,5-diphenylcarbazide (DPC) method (Kathiravan et al. 2011).
The percent decrease in Cr(VI) was determined in accordance with the formula below.
% Cr(VI) reduction: ((Ci - Cf)/Ci) * 100
Ci: initial Cr(VI) concentration mg/L
Cf: final Cr(VI) concentration mg/L
Bioremoval Of Cr Analysis
In order to determine the accumulation of metal on the cell surface, 1 ml of 50 mM EDTA (Merck) solution was applied to the cells precipitated after centrifugation in the previous step. The samples were centrifuged and the supernatant was taken into another tube for ICP MS analysis. The total amount of Cr in the filtrates was analyzed in the ICP-MS device. To determine the amount of metal removed by the strains, the formula given below was used (Anusha and Natarajan 2020).
% of Remove of Cr: ((Ci-Cs)/Ci) * 100
Cr in supernatant (Cs): Remaining concentration of Cr after bioaccumulation (mg/L)
Initial Cr concentration (Ci): Initial concentration of Cr added into the medium (mg/L)
Sem, Stem And Edx Analyses
SEM analysis was conducted to view morphological changes on the surface of Cr(VI) treated cells. STEM (ZEISS, Germany) was used to determine the metal distribution inside bacteria. MT2A cells were grown in a LB medium treated and untreated Cr(VI) (25 mg/L) at 150 rpm at 37°C for 24 hours. The cells were then centrifuged at 6000 rpm for 20 minutes at 4°C, the liquid part was discarded and washed three times with the pellet phosphate buffer (PBS, pH 7.2). Then the pellet was fixed to 2.5% of glutaraldehyde for 4–6 hours at 4°C. Cells were washed with PBS and treated with 1% osmium tetraoxide for 1–2 hours. After fixation, the cells were washed again with PBS and dehydrated by increasing concentrations of ethanol (15, 30, 60, 90 and 100% v/v). The fixed samples were critically dried thoroughly and gold-platinum (QUORUM) coated. The images of the samples were taken using SEM and Energy Dispersive X-Ray (EDX) (ZEISS & EVO LS 10, Germany) devices. By making minor changes in the SEM analysis protocol for STEM analysis, samples of MT2A cells that were Cr(VI) treated and non Cr(VI) treated cells were prepared.
Ftir Analysis
FTIR (Perkin Elmer 400 FT-IR/FT-FIR spectrometer) was performed to detect Cr(VI) binding functional groups on the MT2A cell surface. Cr (VI) (25 mg/L) and Cr (VI) untreated MT2A cells were incubated for 24 hours. The samples were then centrifuged at 5000 rpm for 20 minutes at 4°C. The supernatant was discarded and the pellet was lyophilized after washing twice with PBS (Karthik et al. 2017). Samples were analyzed on the FTIR Spectrometer between 400 cm− 1 and 4000 cm− 1 wavelength.