Our PQQ plasmid consist of five genes: pqqA, pqqB, pqqC, pqqD, pqqE.These are necessary for PQQ synthesis.Built
We modify pet28a plasmid by adding an f1 ori and replacing the T7 promoter with the PR promoter, so that the PQQ gene can be expressed in Bacillus subtilis.Then we test the gene express by the following experiments.
Plasmid Extraction and PCR
We conducted plasmid extraction and PCR to verify that the PQQ plasmid was successfully transferred into Bacillus subtilis RM125 (Figure 5).
The OD600 value of Bacillus subtilis with PQQ plasmid is higher than wild type Bacillus subtilis in log phase and stationary phase. PQQ can improve the efficiency of nutrient utilization of Bacillus subtilis(Figure 6).
Gene Expression On Bacillus subtilis RM125/PQQ
Symbiosis assay of Arabidopsis
Comparing with Arabidopsis which didn’t be inoculated (CK), the leaves area of Arabidopsis thaliana inoculated Bacillus subtilis RM125 with PQQ Plasmid (RM125/PQQ) increased by 1.5 times and capsule number raised by 2 times. Leaves NO. and stalk height also grew significantly. Bacillus subtilis RM125 with PQQ Plasmid can improve both vegetative and reproductive growth of Arabidopsis (Figure 7B and 7C).
In addition, the value of leaf number, leaves area, stalk height, and capsule of Arabidopsis inoculated with our engineered bacteria Bacillus subtilis RM125/PQQ (RM125/PQQ) is higher than the Arabidopsis inoculated with Bacillus subtilis RM125 (RM125), and also more homogenous (Figure 7E and 7F).
Symbiosis assay of Oryza sativa
Oryza sativa seeds were dehulled and sterilized, and constantly shaked in water under darkness for one day then sewed in Murashige & Skoog medium. Thirteen days after inoculation, we found that the value of leaves width, leaves length, shoot height of seedling inoculated Bacillus subtilis RM125 with PQQ plasmid (RM125/PQQ) was higher than the seedling inoculated with Bacillus subtilis RM125 (RM125), and the fresh weight was doubled (Figure 8).
In test on Arabidopsis and Oryza sativa, we got the same result: Bacillus subtilis RM125 inoculated with PQQ plasmid can effectively improve plant growth, and the PQQ gene may be the key factor.
Re-isolate Bacillus subtilis RM125/PQQ
The persistence and effectiveness of the end product might be affected by the success of in planta endosymbiotic Bacillus subtilis RM125/PQQ. Thus, we re-isolated the strain after inoculation to check the endosymbiosis of the artificial endophyte, which may be the most intuitive way for the evaluation. For the Bacillus species, selective medium and 16S rDNA amplicon sequencing checks were used; for PQQ biosynthesis genes, plasmid size, PQQ biosynthesis genes size, and the follow-up Sanger-sequencing of these genes checks were performed (Figure 9 & 10). Taken all together, our results directly indicate the success of Bacillus subtilis RM125/PQQ in planta endosymbiosis.
Inoculate Supernatant of Bacillus subtilis RM125/PQQ
Verification of our assumption that PQQ was secreted off to the supernatant needed to be reassured. We decided to adjust the easiest way, observing the difference after the inoculation by supernatants. Supernatants from Bacillus subtilis RM125 with PQQ plasmid(RM125/PQQ) and Bacillus subtilis(RM125).
In this experiment, we pelleted down the cell and filtered the supernatant. The dilution fold of the supernatant is equal to the value of OD600 value because assumption was made that the amount of metabolites is proportional to the OD600 value.
Results showed that the supernatant of Bacillus subtilis improved the vegetative and reproductive growth of Arabidopsis, and the vegetative growth of Arabidopsis inoculated with our engineered Bacillus subtilis(RM125/PQQ) was slightly higher than Arabidopsis inoculated with Bacillus subtilis RM125 (Figure 11).