Plasmid construction and validation

Firstly, the upstream and downstream about 500 bp respectively of phaC2 gene were used as UP arm and Down arm to construct ‘Up-Arm phaC2 + Down-Arm phaC2’ fragment, and Gibson assembly was used to introduce the composite fragment into vector pJQ200SK linearized by SmaI monoenzyme cleavage, and the recombinant plasmid was introduced into DH5α using the method of transformation, and coated onto gentamicin-resistant LB solid medium, incubated at 37°C overnight and then subjected to colony PCR using the universal primer M13F/R (Figure 1-1-1).

Colony PCR proved that the recombinant plasmid was successfully introduced, and the correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking condition overnight, and the plasmids were extracted and sent for sequencing on the second day, whose results proved that the plasmids were constructed correctly and successfully.

Triparental mating and validation

The verified correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking conditions and cultured, triparental mating was performed using Helper, Sinorhizobium fredii CCBAU45436, and two screenings were performed. The single colonies obtained from the second screening were subjected to colony PCR utilizing the universal primer M13F/R (Figure 1-2-1).

Colony PCR demonstrated that the UA and DA portions of the plasmid underwent homologous recombination with Sinorhizobium fredii CCBAU45436, and the phaC2 gene was successfully deleted. Notably, the ∆phaC2 mutants showed different phenotype from the wild type. The colonies were seen to be distinctly yellow when the single colony were re-picked and streaked on TY solid medium (Figure 1-2-2).

Plant system validation

The deletion of phaC2 resulted in obvious phenotype changes in Sinorhizobium fredii CCBAU45436, and we subsequently inoculated the mutant on Jidou 17 to test whether its original nitrogen-fixing function had been altered. After 15 days of incubation, photos were taken to record the above-ground phenotype, the below-ground phenotype (Figure 1-3-1), and the rhizomatous section (Figure 1-3-2).

There were no significant differences between wild-type and mutant-inoculated plants on phenotype; while mutant strains showed darker color for rhizomatous sections. Their chlorophyll content and dry weight of above-ground parts were examined (Figure 1-3-3).

Analyzing the data of chlorophyll content and dry weight of above-ground parts after 15-day growth, none of the three groups, blank control, wild type and mutant, showed significant differences, indicating that it was not possible to prove whether the differences occurred or not under 15 days of growth. Therefore, a second round of plant experiment was carried out and verified after 21 days of culture after inoculation (Figure 1-3-4).

Chlorophyll content and above-ground part dry weight data from the second round of plant experiments demonstrated that the ∆phaC2 mutant does not affect the growth of the above-ground part of Jidou 17.

Electron microscope scan

To further observe the morphology of the mutant strain, we chose the bacterial solution of the platform stage for transmission electron microscopy observation and counted the number of its PHB particles, whose results are shown in Figure 1-4-1 and Figure 1-4-2.

The number of PHB particles in the wild-type strain and the mutant strain showed a significant difference, further validating the success of the phaC2 deletion module.

Total Lipid TLC Analysis

In order to analyze the changes in fatty acid content in ∆phaC2 strains, we used thin-layer chromatography to analyze the amount of triacylglycerol (TAG) in ∆phaC2 strains as a proxy for fatty acid content (Figure 1-5-1).

∆phaC2 strains were able to show a significant increase in the content of TAG compared to the wild type. We believed that this is due to the altered fatty acid content. This is in line with our design. The deletion of phaC2 gene leads to an increase in the substrates for fatty acid synthesis, which in turn leads to an increase in fatty acid content.