First of all, to promote plant growth, IAA is needed so we researched about several different pathways to IAA.

Our final decision is to use the IAM pathway by converting trypophan into IAA in two steps because it has been shown to work in E. coli in and consists of only two enzymes.[1]Through these two steps, tryptophan is converted via the IAM pathway to indole-3-acetic acid (IAA), a natural plant auxin.Derived from Pseudomonas savastanoi, this pathway involves only two enzymes (IaaM and IaaH) to produce IAA.

In detail,the IAM (Indole-3-Acetamide) pathway is a two-step pathway to produce indole-3-acetic acid (IAA) from the precursor tryptophan. In this two-step pathway, tryptophan is first converted to IAM via tryptophan-2-monooxygenase (IaaM), which is encoded by the iaaM gene.In the second step, IAM is converted to IAA by IAM hydrolase (IaaH), which is encoded by iaaH [2].

We used the pET23b vector, which carries the pT7 promoter and is therefore transformed into E. coli Rosetta (the bacterium is engineered to carry a bacteriophage RNA polymerase that recognizes the pT7 promoter). Since pET23b does not have a LacI inhibitory protein, downstream genes can be transcribed without IPTG induction.

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.

According to the experiment, we learnt that the control strain E. coli Rosetta itself also produces IAA, indicating that E. coli itself also has an endogenous IAA production pathway. Howerver, we found out about a new problem--the environment where the seed will be grown may have low soil EPS, therfore EPS is required provides the necessary substrate for polysaccharide synthesis, the survival of microorganisms and adaptation to the soil environment.

Extracellular polymeric substances (EPS) play a key role in bacterial soil crust, as they can provide microbial adhesion to soil particles, promoting microbial settlement and growth on the soil surface. These aggregates are of great significance for the survival of microorganisms and adaptation to the soil environment.

Therefore, we identified and cloned the galU gene(known to play a key role in enhancing bacterial EPS production), which encodes UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. using the Anthrone-Sulfuric Acid Method in order to to increase EPS production [3].

The GalU gene is known to play a key role in enhancing bacterial exopolysaccharide (EPS) production, so we cloned it into a pET23b expression vector .The recombinant plasmid was transformed into E. coli in competent cells to investigate the effect of GalU genes on the production of EPS when inside E. coil Rosetta.

The measurements include bacterial growth which is monitored by measuring OD600 and glucosamine standard koji was used to assess the EPS concentration in the sample. The EPS concentration is then normalized to OD600 to obtain EPS yield per unit of bacterial growth.

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).

We have solved the problem of the production of auxin and increased the proportion of EPS in the soil but there is still a major issue of the environmental impact of the exposure of the E. coli which contains genes to produce IAA and EPS, disrupting the balance of the content of the soil.

We decided to use arabinose promoter + mazF suicide gene to generate a suicide system so the E. coli will die after seed germinate which would not contaminate the environment.

The reason we choosed the mazF gene is that mazF is the most widely studied and most clearly identified toxin-antitoxin system (TA system). The mazF gene is a downstream gene of the TA system mazEF and encodes a stable toxin protein. MazF is a ribosomal independent mRNA interferase (endonuclease that cleaves single-stranded mRNA at specific sequence sites and is conserved in most microorganisms and some archaeal species).It could result in cell death by restricting the synthesis of protein.

We decided to use the arabinose promoter after we evaluate the function of it through the following experiments.

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 7A. 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 microplate reader and divided by OD600 to obtain the relative fluorescence intensity in Figure 7B .To test the response of the arabic acid promoter to different concentrations of arabic acid, as shown in Figure 7C, 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 7D. 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.

By using an Arabidopsis promoter and suicide gene expression, we have successfully addressed the biosafety concern.

We plan to evaluate the potential promotive effect of IAA-producing engineered E. coli on the germination of various plant seeds. We hypothesize that the IAA produced by the modified E. coli would enhance the germination rates of the selected seeds compared to a control group.

E. coli, engineered to overexpress IAAM and IAAH, were inoculated into 100 mL of M9 medium at a concentration of 100 μg/mL. The culture was incubated on a shaker incubator at 150 rpm for 72 hours at a consistent temperature of 37℃. Following incubation, the culture was centrifuged at 10,000g for 2 minutes. The bacterial pellet was discarded, and the IAA-rich supernatant was collected for seed treatment.

Plant seeds were divided into two groups. The test group seeds were soaked overnight in the supernatant from the engineered E. coli, while the control group seeds were soaked in distilled water. Post-soaking, seeds were placed in cultivation trays, ensuring proper contact and moisture. After three days, germination was observed, and the number of germinated seeds was recorded for both groups. The cumulative germination rates for both groups were calculated and compared to deduce the impact of the engineered E. coli's IAA production on seed germination.

For pine willow seeds, the test group exhibited an 88% germination rate, while the control group had a 76% rate. Similarly, the soybean seeds in the test group had a 90% germination rate, in contrast to the control group's 74%. These findings underscored the positive influence of IAA from engineered E. coli on seed germination. However, challenges arose with wheat, barley, and triticale seeds, as neither group showed the expected germination. This anomaly indicated potential issues in the cultivation protocol. To refine the process and ensure more accurate outcomes in future experiments, a comprehensive optimization of the plant cultivation protocol is planned. Every step, from growth medium selection to optimal cultivation conditions, will be re-evaluated to guarantee improved results in subsequent trials.

In this study, we try to assess the impact of E. coli, specifically engineered to overexpress IAAH and IAAM, on the growth patterns of Arabidopsis thaliana. We posited that the supernatant from this modified bacterial strain could potentially bolster the growth of Arabidopsis thaliana when compared to control group.

After autoclaving, the MS agar medium was cooled to approximately 50°C. Subsequently, 10% (v/v) of the supernatant from the IAA-producing E. coli was incorporated to prepare the MS solid medium enriched with the bacterial supernatant. 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). Simultaneously, a control group (CK group) was established, where seeds were sown on standard MS solid growth medium.

The sown plates were incubated at room temperature, adhering to a 12-hour light-dark cycle. To ensure the robustness of the experimental results, each treatment was replicated thrice. On the 7th day, seedlings were isolated using sterile tweezers, and their root lengths were measured with precision using a calibrated ruler. All measurements were systematically documented. The acquired data was processed using Graphpad Prism software, represented visually as bar graphs. An unpaired student's t-test was employed to discern the statistical significance between the test and CK groups.

Table 2. Arabidopsis photos and root length statistics

The experimental results showed that the root length of the experimental group increased by 2.54 cm on average, while that of the CK group was 1.72 cm, which indicated that the supernatant of the engineered strain had a positive effect on the growth of Arabidopsis thaliana. hese findings pave the way for further exploration and optimization of the use of IAA-producing bacteriA in enhancing plant growth. In the future, we consider extending the experiment to study the long-term effects of IAA producing E. coli on plant growth. Further research into the mechanism of action of IAA and its practical applications in areas such as agriculture and ecological restoration will help to better understand the potential impact of this research.

[1]Spaepen S et al. (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. Federation of European Microbiological Societies Microbiology Reviews 31: 425–448.

[2]Gang, S., Sharma, S., Saraf, M., Buck, M., & Schumacher, J. (2019). Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiellaby LC-MS/MS and the Salkowski Method. Bio Protoc, 9(9), e3230. https://doi.org/10.21769/BioProtoc.3230

[3]https://journals.asm.org/doi/10.1128/AEM.68.2.784-790.2002