For the vector construction, SQR gene was cloned into pSyn_6 vector, and through gel electrophoresis was checked the gene assembly. Figure 1 illustrates the bands of cloned pSyn_6 (5577 bp) in 1-4 wells between 3 and 4 ladder bands, which confirms the success of gene assembly. Also, cloned pSyn_6 plasmid was tested on a presence of SQR gene by PCR amplification using SQR primers. In Figure 2, we can see SQR bands (1271 bp) between 8 and 9 ladder bands that evidences of SQR presence in cloned pSyn_6 plasmid.
Figure 1. Agarose gel electrophoresis (1%) of cloned pSyn_6 plasmid with SQR (5577 bp).
Figure 2. Agarose gel electrophoresis (1%) of PCR amplified products using SQR primers to test its presence in cloned pSyn_6.
After the gene construction, all attention was focused on transformation of cyanobacteria with SQR gene
Figure 3. Lamp with high light intensity.
Figure 4. Cyanobacteria colonies in BG-11 agar plates with spectinomycin.
Confirmation of integration of the cloned pSyn_6 plasmid into genome was done by colony PCR amplification using SQR primers. In Figure 5, we can see colonies with inserted SQR genes, which were used to get liquid genetically modified cyanobacteria culture.
Figure 5. Agarose gel electrophoresis of colony PCR amplified products using SQR primers.
We inserted Sulfide Quinone Reductase into the pSyn_6 сyanobacterial protein expression vector, which performs the function of converting toxic hydrogen sulfide into elemental sulfur. Firstly, we tested the survivability of our cyanobacteria in different concentrations of Na2S and oil. Secondly, to check the functionality of the SQR gene in cyanobacteria we conducted the assay of Na2S reduction.Survival test of cyanobacteria in the oil
Na2S used in the experiments was impure. It was obtained from the industrial waste provided by the MKA engineering company. According to their data, Na2S content was at least 60% and the rest were impurities including heavy metals. The stock solutions of sodium sulfide were prepared assuming that the weight percent of 60%. We conducted survival test of genetically modified and wild-type cyanobacteria in Na2S. On the 2nd day of the experiment, genetically modified cyanobacteria were identified to be more tolerant to the toxicity of Na2S in the solution since the wild-type strain started to show a decrease in the growth in the 500 μM and 1 mM Na2S solutions (figure 6).
Figure 6. Survival test of cyanobacteria in different concentrations of Na2S
The quantitative assay was performed with two concentrations of Na2S (0.5mM and 1mM) using Nanodrop 8000 UV-Vis spectrophotometer, which was set up at the absorbance of 230 nm . The measurements were done before pH adjustment and after pH adjustment to 12. Measurements with pH-adjusted samples were needed to be done in order to minimize the effect of industrial waste content on the results of the assay. The impure Na2S solution contains various metals, which might unfavorably react with SH-. Increasing the pH may reduce the negative effect of metals, reacting predominantly with OH-, rather than creating the extra binding to SH-.
Figure 7 shows the similar absorbance at 230 nm in SQR- and SQR+ strains, however, further measurements indicated a considerable decrease of absorbance at 230 nm in SQR+ strain. The ion transport within the cells could explain the irregular trend of SQR- samples. As for the figure 8, there is no clear difference between SQR- and SQR+ cultures, which can be explained by the impact of the industrial waste composition.
Figure 7. Absorbance values at 230 nm in 500 μM Na2S at pH 12.
Figure 8. Average absorbance values at 230 nm in 500 μM Na2S at not adjusted pH
In order to confirm the work of SQR and show the greater dynamics of bisulfide reduction, our team decided to increase the concentration of sodium sulfide and conduct the assay at 1 mM Na2S.
As shown in Table 1, SQR- strain and SQR+ strain without Na2S do not show any peaks at 230 nm, whereas the positive control (Bg11 with Na2S) demonstrated the high peak at the same absorbance. Therefore, the presence of bisulfide could be indicated by the measurement at 230 nm.
The assay demonstrated higher dynamics of bisulfide reduction during the experiment with 1 mM. Figure 9 and 10 illustrate the considerable reduction of bisulfide in SQR+ in both conditions. While, the SQR- strain indicated the fluctuating measurements.
Figure 9. Average absorbance values at 230 nm in 1 mM Na2S at pH 12
Figure 10. Average absorbance values at 230 nm in 1 mM Na2S at not adjusted pH
Both assay cultures (SQR- and SQR+) had similar initial OD750. Difference between initial and final (2 hours) OD750 measurements of both samples indicate a different effect of Na2S on SQR+ and SQR- cultures. As shown in Table 2 the presence of Na2S in SQR+ sample does not inhibit the growth of cyanobacteria that is supported by the positive average difference in OD750 in table 2. Meanwhile, the OD750 of SQR- strain decreased.
Since the main goal of the project is the bioremediation of oil wastewater, we tested the survivability of genetically modified and wild-type strains of cyanobacteria in different concentrations of oil. The samples were constantly shaken on the agitator. After 3 days since the addition of oil (Figure 11-16), it was identified that cyanobacteria with SQR had higher survival in 0.1%, 0.5%, and 1% oil solutions compared to the wild-type strain.
Figure 11. Survival test of cyanobacteria in 0% oil after 3 days. Control.
Figure 12. Survival test of cyanobacteria in 0.1% oil after 3 days of incubation.
Figure 13. Survival test of cyanobacteria in 0.5% oil after 3 days of incubation.
Figure 14. Survival test of cyanobacteria in 1% oil after 3 days of incubation.
Figure 15. Survival test of cyanobacteria SQR-
Figure 16. Survival test of cyanobacteria SQR+
As seen in Table 3, the survival of cyanobacteria differs between the wild-type and transformed cyanobacteria. The OD750 measurements indicate that growth is impaired in SQR- cultures. Cyanobacteria undergo survival issues to a greater extent with increasing oil content compared to the control. Although SQR+ culture demonstrates a similar trend, the decline in OD750 with an increasing oil concentration is not significant. On the contrary, SQR+ culture has shown to grow faster in 0.1% concentration compared to the control. These results imply that SQR+ are more competent to live in oily conditions with an optimal concentration of 0.1% oil content.
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