Team:NCHU Taichung/Result

Result

Overview

The primary goal of our project is to achieve circular agriculture by reuse of Oryza sativa straws. Decomposing agricultural wastes accompanished by the Oryza sativa becomes the first aim. To fulfill the aim, we transferred the cellulase genes into Bacillus subtilis and produced enzymes with scaffolding protein that could be assembled as a cellulosomal enzyme system to degrade Oryza sativa straws.

Construction of Cellulosome

On the gel electrophoresis diagram, the third well contains the plasmid of scaffolding protein gene cipA. From seventh well to twelfth well, they respectively contain the plasmid of cellulase gene celA, celK, celR, celS, xynC and celZ (Figure 1).

These cellulase can be classified into three groups. CelA and CelR are endo-glucosidases. CelK and celS are exo-glucosidases. XynC and xynZ are both xylanase. In order to achieve the maximum efficiency, the cellulase that have better activity in each group are chosen for the experiments, the construction of our cellulosome containing XynC, CelK, CelA and scaffolding protein CipA.

Figure 1 Cellulase plasmid extraction

Sugar concentration quantification

To quantify the sugar concentration in our broth, we mixed the DNS with the sugar that will change color while reaching one hundred celsius degrees. After calibration and testing of the OD₆₀₀ value, we obtained the concentration of the sugar in the Oryza sativa straw by analyzing how much the sugar generates, and calculated the weight of sugar generated from Oryza sativa straws by concentration.

Our cellulosome are assauredly viable because ensurement is made that our experimental environment is sterile and only the cellulosome we put in can decompose the cellulose and hemicellulose (Figure 2).

Figure 2 Sugar concentration quantification

The next step is to transfer the pyrroloquinoline quinone (PQQ) synthesis genes into the Bacillus subtilis, which can make the Bacillus subtilis produce pyrroloquinoline quinone and act as endophyte to accelerate Oryza sativa growth.

Extract PQQ plasmid from Bacillus subtilis WB800

Figure 3 PQQ plasmid extraction from Bacillus subtilis WB800 (Plasmid didn’t cut by restriction enzyme,so the circular DNA will locate above 10000bp).

Gene Expression On Bacillus subtilis WB800

To test the effectiveness of PQQ on plants, we inoculate Bacillus subtilis WB800 within or without the PQQ plasmid into Arabidopsis.

In the picture below, respectively shows phenotypes of 5-week-old Arabidopsis inoculated with Bacillus subtilis WB800 with PQQ plasmid (PQQ), with Bacillus subtilis WB800 (WB800), and with water (CK) (Figure 4A).

Bacillus subtilis WB800 with PQQ plasmid significantly improved stalk height, the stalk height of Arabidopsis with PQQ is about 8 cm, and the stalk height of Arabidopsis with Bacillus subtilis WB800 is about 2 cm (Figure 4B).

Arabidopsis inoculated with water has one primary inflorescence. Arabidopsis inoculated with Bacillus subtilis WB800 have 3 inflorescences, including primary and secondary inflorescence. Arabidopsis inoculated with Bacillus subtilis with PQQ plasmid have nearly 5.25 for average inflorescences, encompassing primary, secondary and tertiary inflorescence (Figure 4C).

Bacillus subtilis WB800 with PQQ plasmid possibly help plants to reach reproductive stages earlier.

Arabidopsis inoculated with PQQ has the highest performance in the leaves area of Arabidopsis. The leaves area is about 5 cm², in contrast to 4 cm² of Bacillus subtilis WB800, and to 3 cm² of CK (Figure 4D).

Figure 4 Bacillus subtilis WB800 with PQQ plasmid possibly help plants to reach reproductive stages earlier.

However, we discovered that WB800 has a kanamycin resistance gene in its genome(as knockout strain marker). So we decided to choose Bacillus subtilis RM125 as the new host.

Engineering success

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).

Figure 5 PQQ plasmid extraction and PCR.

Growth Curve

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).

Figure 6 The OD₆₀₀ value ofBacillus subtilis with PQQ plasmid is higher.

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).

Figure 7-1 Artificial endophyte Bacillus subtilis RM125/PQQ promoted Arabidopsis growth. Figure 7-2 Artificial endophyte Bacillus subtilis RM125/PQQ promoted Arabidopsis growth.

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).

Figure 8 Artificial endophyte Bacillus subtilis RM125/PQQ promoted Oryza sativa growth.

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.

Endophyte Re-isolation

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.

Picture 1. Artificial endophyte.(A)Bacillus subtilis RM125/PQQ re-isolated from Arabidopsis.(B)Bacillus subtilis RM125/PQQ re-isolated from Oryza sativa.

Figure 9 Plasmid extraction of Bacillus subtilis RM125/PQQ re-isolate from plant.(Plasmid didn’t cut by restriction enzyme,so the circular DNA will locate above 10000bp) Figure 10 PCR result of Bacillus subtilis RM125/PQQ re-isolate from plant

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).

Figure 11-1 Supernatant of Bacillus subtilis subtilis RM125/PQQ slightly improved the growth of Arabidopsis. Figure 11-2 Supernatant of Bacillus subtilis RM125/PQQ slightly improved the growth of Arabidopsis.

Proof of concept

Culture Bacillus subtilis RM125/PQQ with saccharified straw liquid

Our final purpose is to turn the agriculture waste to the medium of benefical endophyte, and use the bacteria with the medium as a growth biostimulant to increase agricultural productivity. The final goal is to find the best carbon-nitrogen ratio of the saccharified straw liquid to culture Bacillus subtilis RM125 with PQQ plasmid. We have Oryza sativa straw for carbon sources and Okara for nitrogen sources. After a series of experiments, we found that 1:4 is the best ratio of Oryza sativa straw to Okara(Figure 12). The OD600 value of Bacillus subtilis RM125 with PQQ can achieve nearly twice or twice and a half increase in the saccharified straw liquid (Figure 13).

Figure 12 the best carbon-nitrogen ratio of the saccharified straw liquid to culture Bacillus subtilis RM125 with PQQ plasmid. Figure 13 The OD600 value of Bacillus subtilis RM125 with PQQ can be about 1 to 1.5 in the saccharified straw liquid.

Oryza sativa Inoculation

To avoid Soil-Borne disease, seeds of Oryza sativa（Oryza sativa var. Japonica Taiken 9) was sterilized by NaClO, sowed in potting soil, and inoculated three times (12 days, 33 days, 60 days after sowing_DAS).

For biosafety reasons, we filtered out the recombinant Bacillus subtilis, inoculated Oryza sativa only with supernatant. After culturing our endophyte in saccharified straw liquid,we adjusted the OD600 value to 1, then pellet down the bacteria and filtered the supernatant as stock inoculant.The stock inoculant was diluted 100 times 200times and 500times.The concentration of the supernatant is defined as 1x, 1/100x,1/200x, and 1/500x.

Picture 2. The phenotype of Oryza sativa inoculated with Supernatant of Bacillus subtilis RM125/PQQ (46 days after sowing). Picture 3. The phenotype of Oryza sativa inoculated with Supernatant of Bacillus subtilis RM125/PQQ (54 days after sowing).

Supernatants of higher concentration seemed to be more effective (Picture 2 & 3 ). Treatment with 1x and 1/100 x supernatant significantly increased the leaves number, shoot height, and leaf length (Figure 14A,14D,14E).The tiller quantity of Oryza sativa inoculated 1x supernatant escalated by 50.3%(Figure 14B) and the dry weight was doubled (Figure 14C). In the meantime, the tiller quantity of Oryza sativa inoculated 1/100x supernatant was raised by 29%(Figure 14 B), the dry weight was increased by 96%( Figure15C), and chlorophyll content raised 1.5 times (Figure 14F).

More tillers means the Oryza sativa may have more ears ,and the increase of chlorophyll content means the increase of photoassimilates.All these phenomena pointed out that that inoculant can improve Oryza sativa growth and have a great opportunity to increase the yield.

Figure 14 Supernatant of Bacillus subtilis RM125/PQQ cultured in saccharified straw liquid can promote Oryza sativa growth. Figure 15 Supernatant of Bacillus subtilis RM125/PQQ cultured in saccharified straw liquid can increase the tiller quant and the clum diameter of Oryza sativa Figure 16 Oryza sativa phenotype measurement.

The vegetative phase of Oryza sativa is about two months, and about 3-5 seedlings will be planted together until the total amount of tiller reaches 20-25, then the Oryza sativa is ready to enter the reproductive phase. According to this, 4 tillers is the lowest value for every single plant. While the control groups have about 4 tillers, inoculated Oryza sativa(1x,100x) have already reached 6-8 tillers,(Figure16B) which means that inoculants shorten the time for Oryza sativa to reach 4 tillers, helping Oryza sativa enter the reproductive phase earlier. Water sources are becoming a serious problem in agriculture because of global climate change. If the time for Oryza sativa cultivation shortens, a lower amount of water is needed.

Besides, the culm diameter also increased, which means that the inoculants may have the potential to improve breaking-type lodging resistance (Figure 15C).

Harvest is just around the corner!

The treatment group is almost ready to harvest, and the experiment is still in progress.

• nchutaichung2021@gmail.com
• NCHU iGEM Team
• igem_nchu_taichung
• NCHU_Taichung
• NCHU, Taichung, Taiwan