Difference between revisions of "Part:BBa K5015001"

Latest revision as of 15:21, 9 October 2023

IAAH and IAAM can promote the synthesis of growth hormone indoleacetic acid by Escherichia coli

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.

Usage and Biology

We used the plasmid pET23b as the basis for two separate genes, iaaM and iaaH, downstream of the pT7 promoter. The iaaM enzyme transforms tryptophan into Indole-3-Acetamide (IAM), then the IaaH enzyme transforms IAM into IAA, our final product. After sequencing, we transformed the said plasmid into the E. Coli Rosetta strain.

Figure 1. The design of iaaM and iaaH.

Figure 2. Gel electrophoresis of the iaaM and iaaH.

Characterization

The engineered E. coli 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 3, which indicate that the overexpression of both genes significantly increases the production of IAA.

Figure 3. Comparison of IAA production.

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.

The observed increase in root length in the test group, with an average of 2.54 cm compared to 1.72 cm in the CK group, suggests that the supernatant from the engineered bacterial strain positively influences the growth of Arabidopsis thaliana. This significant enhancement in root length indicates the potential benefits of utilizing the products from the engineered strain in promoting plant growth.

Potential application directions

Our experiment proves the viability of engineering E. Coli into producing extra IAA for plant usage. As our world progresses towards more frequent, easier genetic engineering processes, artificial IAA production can be allowed and improved easily, which indicates a more efficient, harmless pathway for plant growth since IAA is a natural hormone and not a synthetic one.

Sequence and Features