This research was carried out in the Biotechnology Laboratory of the School of Agricultural and Environmental Sciences of the Pontificia Universidad Católica del Ecuador Sede Ibarra.
Elaboration of the biosensor
For the elaboration of the biosensor, the sequence of the chr gene that gives the bacterium resistance to chromium and the sequence of the lux operon to generate the optical response of the biosensor was searched in the registry of community collection of genetic sequences available in iGEM (International Genetically Engineered Machine) to later assemble the plasmid in the Biotechnology company "Humanazing Genomics © MAROGEN Inc.". The result being the genetic synthesis described in Table 1 (MACROGEN Inc, 2018).
The result of the assembly of the gene sequences produced a plasmid with a size of 1,847 base pairs (bp) and a guanine and cytosine (GC) percentage of 60.23%.
For the modification of the genes of the E. coli bacteria, the Electroporation method described by Bastida and Levi (2019) was used, where a Thermo Scientific ULT1230A freezer was used to prepare the competent cells, since these must be preserved at a temperature of 4°C. After this, 10 µl of the manufactured plasmid plus 20 µl of the competent cells were taken and added in a 2 ml eppendorf tube, this mixture was centrifuged and placed in the freezer again.
For the transformation of E. coli, the Electroporation Neon Device equipment was set at 2500 volts for 4 milliseconds and two pulses for the transmutation of E. coli (Gómez and Garns, 2003). To prepare the Electroporation equipment, the Neon Pipette Station was connected in which the Neon Tube cuvette was inserted with 3 ml of electrolytic buffer (Electrolytic Buffer) with a temperature of 2°C to 4°C (Gómez and Garns, 2003).
LB Broth Base broth was prepared in a 250 ml boeco bottle, according to the manufacturer's instructions. Next, 10 ml of this mixture was dispensed into 24 test tubes and sterilized with the help of the ICAN CLAVE autoclave for 40 minutes. Having the 24 test tubes already sterilized, hexavalent chromium concentrations of 0.05 mg l−1 were placed; 0.1 mg l−1; 0.2 mg l−1; 0.4 mg l−1; 0.8 mg l−1 and 1.0 mg l−1.
Next, 100 µl, equivalent to 2x10 6 CFU of the transformed cells, was added to each test tube, incubated for 2 hours and chromium analysis was carried out by the EPOCH Microplate Spectrophotometer method, where 100 µl was added to the cells. samples that were previously incubated, placing the different chromium concentrations in each column of the microplate and 5 repetitions in each row. With the help of the EPOCH Microplate Spectrophotometer from Biotec Instruments and a computer program for data analysis Gen5 Software, the absorbance analysis of the samples was carried out to obtain the calibration curve (Macías et al., 2019).
Analysis of the process for the detection of chromium using a modified E. coli strain.
The E. coli bacteria were seeded in quantities of 2x106 CFU genetically transformed in test tubes with LB Broth broth from the manufacturer LAB a Neogen Company (Luria Bertani) ®, a medium that does not intervene in the reaction between the bacteria and the metal and provides the necessary nutrients for the bacteria, plus the transition metal, verifying their resistance and the optical response of the luciferase gene and the interaction between the metal and the medium (García et al., 2005).
For the analysis of the biosensor to detect chromium, liquid samples (Figure 1) were qualified with hexavalent chromium at known concentrations of 0.05 mg l−1; 0.1 mg l−1; 0.2 mg l−1; 0.4 mg l−1; 0.8 mg l−1 and 1.0 mg l−1 ; indicating the presence of the metal before the different levels of luminosity directly proportional to the concentration of the toxic agent, this being the higher the concentration of metal, the more intensity of the luminescence.
In the research carried out by Huelves (2008), the enzyme that catalyzes the luminescence reaction is encoded in the enzymatic protein of luciferase, an operon that was modified together with the chromium resistance genes by inserting into the E. coli bacteria, thus obtaining a medium that translates into light emission.
Field application of the two methods to quantify hexavalent chromium
For the field application of the two methods used to quantify hexavalent chromium, the research of Jurado et al. (2017). The first method suggested uniformly sectioning the total length of the river, which consisted of 12.71 km, identifying accesses, the presence of contamination by wastewater discharges (Figure 2) and agricultural activities, a method applied to date in the monitoring of river samples.
Two levels were raised; the macro location that determined the most representative sections according to the total length of the Pichaví river; the micro-location that implied the equivalent division of the total length of the river, with a distance of 2 km between each point (Figure 3), a distance that must be considered according to the different methods applied in the field for the collection of a sample, due to the estimation of the total uniform mixture of water between each section, thus generating a total of 6 points for the collection of water samples (Jurado and Mercado, 2017).
In the research, a statistical design of paired plots was applied by applying the t-Student test, in order to see the relationship between the methods applied for the measurement of hexavalent chromium and define the hypothesis. For the normality of the data, the Shapiro – Wilk test (Rossiter, 2014) was used. Two tests were used being these cases: the first test used 6 concentrations of hexavalent chromium of 0.05 mg l−1; 0.1 mg l−1; 0.2 mg l−1; 0.4 mg l−1; 0.8 mg l−1 and 1.0 mg l−1; This served to standardize the measurement method and verify that bacteria have the potential to measure chromium. Subsequently, 6 liquid samples were taken from the Pichaví river and the biosensor technique was compared with a commercial and field technique.
To compare the efficiency of the two methods, standard liquid samples and river samples were analyzed by the EPOCH Microplate Spectrophotometer from Biotec Instruments and with the SMART 2 Colorimeter from LaMotte Company, comparing the normality of the results by means of a Shapiro test. - Wilk.