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.

Transformation of S. elongatus PCC 7942 with SQR

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.

Transformation verification

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.

Validation of a Part

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

Na₂S reduction assay

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 [1]. 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.