Team:XHD-Wuhan-A-China/Engineering
Engineering
In order to enhance the denitrification of rhizobia, we designed an enhanced denitrification system.
Enhanced denitrification system
We designed an enhanced denitrification system, which includes two plasmids: napA-pBBR1MCS2 and nirK-pBBR1MCS2. We designed the napA gene circuit J23100-RBS-napA (Figure 1). In order to realize the function of this gene circuit, we assembled this circuit on the pBBR1MCS2 plasmid backbone and connected to amilCP for characterization (Figure 2). At the same time, we designed the nirK gene circuit J23100-RBS-nirK (Figure 3). In order to realize the function of this gene circuit, we assembled this circuit on the pBBR1MCS2 plasmid backbone and connected to amilCP for characterization (Figure 4).
Figure 1. Constitution of J23100-RBS-napA gene circuits
Figure 2. Design of Enhanced denitrification system plasmid (napA-pBBR1MCS2)
Figure 3. Constitution of J23100-RBS-nirK gene circuits
Figure 4. Design of Enhanced denitrification system plasmid (nirK-pBBR1MCS2)
Usage and Biology
We verified the length of the recombinant plasmid by restriction enzyme digestion to ensure the success of the recombinant plasmid (Figure 5). Theoretically, the transformation of napA-pBBR1MCS2 or nirK-pBBR1MCS2 will increase the copy number of napA and nirK genes, increase the content of denitrification enzymes, and improve the denitrification ability of the strain.
We cultivated strains containing napA-pBBR1MCS2, nirK-pBBR1MCS2 and wild-type strain in LB liquid medium. And the content of amilCP was measured by OD588 every 30 minutes, which results are shown in (Figure 6).
Figure 5. Gel electrophoresis of plasmid MCS-napA(nirk)-amilcp
Figure 6. Changes in amliCP concentration of LB liquid medium
We have successfully constructed an enhanced denitrification system and successfully verified the reliability of the system to decompose nitrates. Through our system, we increased the number of copies of napA and nirK successfully. We verified the system that expresses napA-anilCP and nirK-anilCP fusion protein over time, so that the optimal culture time can be determined. Our project can provide new solutions for decomposing excessive nitrate in the soil and improving soil compaction.
