Team:XHD-Wuhan-A-China/Proof Of Concept
Proof of concept
Project and design
Due to large-scale irrational farming and the abuse of chemical fertilizers, soil compaction is caused, which causes changes in soil structure, decreases in porosity, increases in hardness, and decreases in infiltration capacity, resulting in degradation of soil fertility properties. Among them, the abuse of chemical fertilizers leads to obvious salinization and acidification of the soil, which is one of the important reasons for soil compaction. The application of microbial agents is a good way to improve saline soil. In order to increase the denitrification of rhizobia, we designed an enhanced denitrification circuit. By increasing the copy number of napA gene and nirK gene in bacteria, the enzyme activity related to the denitrification process can be increased, thereby enhancing the denitrification effect of rhizobia.
Results
Sinorhizobium fredii HH103 culture
We cultivated Sinorhizobium fredii HH103 in the laboratory and activated Sinorhizobium fredii HH103 by streaking on a solid medium. The results are shown in Figure 1.
Figure 1. Plate streak cultured Sinorhizobium fredii HH103
Enhanced denitrification system
Enhanced denitrification system has four parts: Promoter J23100 (BBa_J23100), RBS (BBa_B0034), napA/nirK (BBa_K3926004/BBa_K3926005), TT (BBa_B0015). In order to enhance the denitrification effect of Rhizobium, we chose the strong promoter J23100 as the promoter of the system to maximize the copy number of the target gene. Both napA and nirK genes are key genes related to denitrification. The backbone we selected is an expression vector that can be expressed in E. coli and Rhizobium (Figure 2).
Figure 2. Design of Enhanced denitrification system
After that, we introduced the enhanced denitrification system we built into the engineered bacteria and coated the plate. After a single colony grew, we found that our engineered bacteria successfully expressed the blue color protein amilCP. Although napA and nirK are both denitrification-related genes, the gene lengths of the two genes are not the same. It was found that the number of gene copies is different in the two genes on the same promoter and the same plasmid backbone. Based on the blue color of the bacterial solution (Figure 4), the copy number of nirK gene is higher than the copy number of napA.
Figure 3. Single colony with enhanced denitrification system
Figure 4. The expression levels of target genes are different after introducing different enhanced denitrification systems
Finally, we verified the denitrification ability of the engineered bacteria in order to explore whether it has real potential for proposed implementation. We put the engineered bacteria in a liquid medium containing additional 0.5mM potassium nitrate for 24 hours, then centrifuged to collect the supernatant, to calculate whether the bacteria contained napA-pBBR1MCS-2 or nirK-pBBR1MCS-2 have the ability to reduce the concentration of nitrate ions in the system. The color reaction of detecting nitrate concentration in strains containing napA-pBBR1MCS2, nirK-pBBR1MCS2 and wild-type strain is shown in Figure 5.
Figure 5. Color change at different nitrate concentrations in liquid medium culturing napA-pBBR1MCS2, nirK-pBBR1MCS2. From left to right: Blank; LB liquid medium; napA-pBBR1MCS2; nirK-pBBR1MCS2.
We used a microplate reader to quantitatively analyze the changes in nitrate concentration after adding engineered bacteria. The smaller the OD550, the lower the nitrate content. The results are shown in Figure 6. The OD550 of the nirK group is smaller than that of the napA group, which is consistent with the previous results.
Figure 6. Changes in nitrate concentration in each group
Conclusion
We successfully constructed two enhanced denitrification systems (napA-pBBR1MCS2; nirK-pBBR1MCS2). These two systems have different abilities to degrade nitrate. The related results are shown in Figure 5 and Figure 6. The function of the two systems are in line with expectations, and they have the potential to be successfully applied to agricultural production to improve soil compaction.
References
Wang, P., Li, S., & Lee, H. K. (1998). Measurement of nitrate and chlorate in swimming pool water by capillary zone electrophoresis. Talanta, 45(4), 657-661.
