Auxin, primarily indole-3-acetic acid (IAA), is an important hormone present in plants that significantly influences plant growth and development. Among these, IAA has a particularly prominent effect on seed germination and root growth. However, the production and application of IAA are influenced by various factors, including difficulties in synthesizing it on a large scale and at a low cost, as well as its potential environmental impact. Our goal is to develop a method that can promote seed growth in ecosystems.

The primary goal of our project is to mitigate the issue of soil loss by increasing plant growth. In the natural world, plant growth is typically stimulated by a hormone called Indole-3-Acetic Acid (IAA), and one key pathway for increasing plant growth is to provide it with extra IAA. To achieve this, we choose to genetically modify our E. Coli cells to produce more IAA than the wild type.The engineered E. coli strings are grown in a Luria-Botani (LB) media at 30 degrees Celsius and 200 rpm shaking. Since IAA easily degrades upon light exposure, the flasks are covered in aluminum foil and the shaker is covered with newspaper to prevent light exposure. We take 1.5mL of bacterial culture every 24 hours and mix the post-centrifuge (10000rpm, 1min) supernatant with Salkowski reagent (2% of 0.5M FeCl₃ in 35% perchloric acid). The absorbance was measured spectrophotometrically at 530 nm with a prepared standard curve, and our results are shown below.After converting the absorbance data into direct concentrations, we can clearly see that at every 24-hour checkpoint, there is a statistically significant difference between IAA production of our transformed strains and wild-type strains. The differences at each standpoint remain relatively consistent at about 65 micromolarity. Interestingly, the results also show that without any genetic engineering, the wild-type strains do still produce a significant amount of IAA by themselves.The results of the expression engineering strain are shown in Figure 2, which indicate that the overexpression of both genes significantly increases the production of IAA.

All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD).

To evaluate the promotive effect of engineered E. coli on plant seed germination, E. coli modified to overexpress IAAM and IAAH were inoculated into 100 mL of M9 medium at a concentration of 100 μg/mL. This culture was incubated on a shaker incubator with 150 rpm for 72 hours, maintaining a consistent 37℃ throughout. After centrifuging at 10,000g for 2 minutes, the bacterial pellet was discarded and the supernatant was collected. Subsequently, the chosen plant seeds were divided into two groups. One group was soaked overnight in the supernatant from the engineered E. coli, while the other group was soaked in distilled water as a control. After soaking, the seeds were placed in cultivation trays, ensuring they made contact with the base of the tray and maintained appropriate moisture. Three days later, the germination of seeds in both groups was observed and the number of germinated seeds was recorded. The cumulative germination rates of the two groups were calculated and compared to assess the impact of IAA produced by the engineered E. coli on plant seed germination. The results indicated that in the test group, the germination rate of pine willow seeds was 88%, while in the CK group, it was 76%. Furthermore, in the test group, the germination rate of soybean seeds was 90%, whereas in the CK group, it stood at 74% (Table 1). This suggests that the engineered E. coli's IAA production positively influenced the germination of these seeds.

Table 1. Plant seed germination rate statistics.

We encountered some challenges in this experiment for reasons that remain unclear. It's possible that due to shortcomings in our cultivation technique or an inappropriate selection of cultivation duration, we observed an unexpected outcome: neither the control group nor the experimental group showed the anticipated germination for wheat seeds, barley seeds, and triticale seeds. This result highlighted potential deficiencies in our current cultivation protocol that require further optimization and adjustment. To ensure the success of future experiments and obtain more accurate data, we plan to comprehensively improve and optimize our plant cultivation protocol. We will re-evaluate every step, from selecting the appropriate growth medium to determining the optimal cultivation conditions, to ensure better results in our next experiment.

We also evaluated the impact of the supernatant from the engineered bacterial strain on the growth of Arabidopsis thaliana. To prepare the MS solid medium containing the bacterial supernatant, after autoclaving, the MS agar medium was cooled to approximately 50°C, and then 10% (v/v) of bacterial supernatant was added. Under sterile conditions, disinfected Arabidopsis thaliana seeds were evenly sown on the MS solid growth medium containing the supernatant using 1 mL pipette (test group). For comparison, we also established a control group (CK) where seeds were sown on regular MS solid growth medium. Subsequently, both sets of plates were placed at room temperature and subjected to a 12-hour light-dark cycle to simulate natural growth conditions. To ensure the reliability of the experimental results, each treatment was replicated three times, and the average values and standard deviations were calculated. On the 7th day after sowing, seedlings were carefully picked using sterile tweezers. The root length of these seedlings was measured using a calibrated ruler. All data points were meticulously recorded for further analysis. The data were visualized in the form of bar graphs using the Graphpad Prism software. To determine the statistical significance between the test group and the CK group, an unpaired student's t-test was conducted. A p-value of less than 0.05 was considered to indicate a significant difference between the two groups.

Table 2. Arabidopsis photos and root length statistics

As the GalU gene plays a crucial role in enhancing bacterial extracellular polysaccharide (EPS) production, we have decided to overexpress galU. EPS plays a key role in bacterial soil crust by providing microbial adhesion to soil particles and promoting microbial colonization and growth on soil surfaces. Additionally, extracellular polysaccharides can form microbial aggregates, which are of great significance for microbial survival and adaptation to the soil environment.The successfully engineered strain will be expanded in culture.At predetermined time points (4, 8, 12, 16 hours), bacterial growth was monitored by measuring the OD600. Concurrently, cells were centrifuged, and the supernatant was used to quantify EPS using the anthrone-sulfuric acid method. In brief, a sample of the supernatant was combined with anthrone reagent, followed by the addition of concentrated sulfuric acid. The mixture was subsequently heated, and the developed color was measured spectrophotometrically at 620 nm. A standard curve, established using known concentrations of a standard polysaccharide, facilitated the determination of the EPS concentration in the samples. The EPS concentration was then normalized to the OD600 to obtain EPS production per unit of bacterial growth. To establish a standard curve, 0-200 mM glucose was used as the standard substance and quantified using the anthrone colorimetric method, with absorbance measured at 620 nm, as shown in Figure 4A. Compared to the control group, the engineered bacteria significantly increased EPS levels, which also increased with time, as shown in Figure 4B. The results indicate that the engineered strain can significantly enhance EPS production.

All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD).

An unavoidable issue when utilizing genetically engineered bacteria strains is contamination, where our strains might accidentally leak out to the outer environment, potentially polluting the soil and impacting the ecology in a negative way. Hence, we designed an apoptosis system under the arabinose promoter sequence with the goal of proper cell death at contact with arabinose. We used the plasmid sample pSB1A3, which contains the arabinose promoter sequence, as the basis for the mazF gene. We then transformed these constructs into th E. Coli DH5a strains with the heat shock method. the mazF gene codes for a series of protein toxin enzymes that severs mRNA and hence inhibit cellular protein expression and consequentially kill the cells.

All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD). Statistical significance between induced and non-induced cultures was determined using a Student's t-test, with p < 0.05 considered statistically significant.)

The mRFP (monomeric Red Fluorescent Protein) gene was utilized as a reporter to assess the functionality of the arabinose promoter. The intensity served as an indicator of the activity of the arabinose promoter，the result is shown in Figure 5A. When the OD600 of the engineered bacteria and the control strain is 0.6, 1 mM arabic acid is added. After induction for 4 hours, the fluorescence intensity is measured using an enzyme-linked immunosorbent assay microplate reader and divided by OD600 to obtain the relative fluorescence intensity in Figure 5B .To test the response of the arabic acid promoter to different concentrations of arabic acid, as shown in Figure 5C, when the OD600 of the engineered bacteria is 0.6, 0.1, 0.5, 1, 2, and 4 mM arabic acid is added. After induction for 4 hours, the fluorescence intensity is measured and divided by OD600 to obtain the relative fluorescence intensity. To induce the expression of MazF, varying concentrations of arabinose were added to the cultures. Bacterial growth was monitored at specific time intervals (i.e., 2, 4, 6 hours post-induction) to assess the inhibitory effects of MazF expression，The results are shown in Figure 5D. In conclusion, it can be concluded that MazF induction has a significant inhibitory effect on bacterial growth compared to the uninduced control group. After 6 hours of induction, bacterial growth was significantly inhibited, and after 14 hours, growth almost completely stopped.