1 Successfully construct two plasmids including FGF21 and P9

Given that we have designed multiple small fragments, including the enhancement peptide LEISSTCDA, LMWP, and FGF21, it’s difficult to have the relevant fragments in existing plasmids. Therefore, we opted for a fully synthetic approach to construct our plasmid.During the synthesis process, we encountered some issues such as stem-loop formation; however, we ultimately obtained a plasmid without mutations through sequencing and successfully completed the transformation.

Figure 1.1.1- The construction of FGF21 plasmid

Figure 1.1.2- The construction of P9 plasmid

Figure 1.1.3- The sequencing result of FGF21 plasmid

FIgure 1.1.4- The sequencing result of P9 result

2 Set up optimal condition for engineering bacteria growth

2.1 Background:

The study of Lactococcus lactis, a significant lactic acid bacterium widely used in the dairy industry for the production of cheese and other fermented products, is crucial for optimizing fermentation processes. Understanding the growth dynamics of L. lactis in culture media is fundamental to controlling and enhancing the efficiency of these industrial applications. Traditional growth models, such as the Malthus model, which assumes a constant growth rate, do not account for environmental limitations like resource availability and space.

In the complex environment of a culture medium, the growth of L. lactis is influenced by various factors including the initial inoculum size and environmental resistance, which is directly proportional to the bacterial population. To accurately simulate the growth of L. lactis, it is necessary to adjust the natural growth rate parameter to reflect the environmental resistance and carrying capacity.

This research focuses on developing a mathematical model that accurately represents the growth of L. lactis in culture media by incorporating these environmental factors and validating the model with experimental data.

2.2 Result:

The experimental data acquisition process was critical for fitting the growth model of Lactococcus lactis. To ensure a precise fit, continuous and sufficient experimental data was necessary. The specific steps undertaken for data collection were as follows:

1.Inoculation A single colony of the Lactococcus lactis strain was inoculated into 5 ml of MSR medium in a culture tube and allowed to grow overnight.

2.Dilution and Culturing: The overnight bacterial culture was diluted 500-50 times in fresh MSR medium at a concentration of 100 μg/ml.

3.Sampling: 200 μl of the diluted culture was then aliquoted into each well of a 96-well plate, with three replicates prepared for each sample.

4.Growth Measurement: A microplate reader and Gen5 software were used to collect growth curve data at 10-minute intervals.

The raw data obtained from the experiment, which included the optical density (OD) readings at 600 nm, were converted into bacterial concentration growth curves. Using a Python program, the OD600nm readings were translated into concentration units (CFU·ml^-1), allowing for the estimation of bacterial numbers.

Figure 1.2- The growth curve of bacteria

3 Successfully induce FGF21 expression

First, we set 15 ng/ml cholate as the low concentration stimulation condition according to the bile acid concentrations provided in the literature. However, since this concentration is relatively low, we established 100 ng/ml as the high concentration group to better simulate the bile acid levels in the human body. The duration was set to 30 minutes to explore the level of the bacteria's responses over a short period.

Figure 1.3.1- The ELISA result under different cholate concentration condition

Compared to the wild-type bacteria, the transformed bacteria produced a smaller amount of FGF21, with only 193 pg/ml secretion under the stimulation condition of 100 ng/ml cholate. Based on our analysis, we believe that this may be attributed to the lower bile acid concentration and shorter stimulation time. We have further designed the following experiments to investigate this.

Figure 1.3.2- The ELISA result with different stimulation time

We found that by extending the stimulation time, the concentration of FGF21 in the supernatant was significantly increased. The FGF21 concentrations at stimulation times of 30 minutes, 60 minutes, and 120 minutes were 206, 663, and 1689 micrograms per μm, respectively, reaching the microgram level. This provides data support for our next experimental plan.

4 Successfully purify FGF21 protein

After centrifuging the bacteria, the supernatant and bacterial solution were collected separately. The bacterial cells were washed twice with TBS and then lysed with lysozyme. (Note: To obtain a high concentration of the FGF21-LMWP fusion protein, we chose to isolate and purify the protein from the bacterial cells rather than the supernatant. ) The lysed bacterial solution is referred to as "Sample” or “Before purification." After column equilibration, we performed loading, washing, and elution steps to obtain the purified solution, referred to as "After purification." SDS-PAGE analysis shows that we obtained a relatively pure FGF21-LMWP fusion protein, which can be used for the next step in validating cell function improvement.

Figure 1.4.1- Diagram of Ni-Affinity Chromatography Steps

5 FGF21 secreted by engineered L. lactis strain NZ9000 improves insulin resistance

To verify the biological function of FGF21 (BBa_K5283016) in improving insulin resistance, we established an adipocyte model of insulin resistance. First, we treated this insulin resistance using FGF21 secreted by engineered L. lactis strain NZ9000. After that we measured the activation levels of downstream pathways after insulin stimulation to reflect the biological function of FGF21.

For the collection of media conditioned by RAW264.7 macrophages (RAW-CM), RAW264.7 macrophages were grown to 90% confluency in DMEM containing 10% FBS. Then, the cells were stimulated with 100 ng/ml lipopolysaccharide (LPS) for 3 h. After stimulation, the cells were cultured in new serum-free DMEM for 24 h. The media was collected, filtrated through a 0.22 μm filter, and used as RAW-CM. Insulin resistance of 3T3-L1 adipocytes could be induced by incubation with RAW-CM. To assess the insulin resistance improvement effects of FGF21, RAW-CM and 200 μg/ml FGF21 purified from engineered L. lactis strain NZ9000 were added into 3T3-L1 adipocytes. After treatment for 24 h, 100 nM insulin was added to activate the insulin-induced PI3K/Akt/mTOR signaling pathway . The insulin sensitivity was detected by the AKT (Ser473) phosphorylation, which was qualified using Western Blot.

Figure 1.5.1- The overall process of verification

Figure 1.5.2- The adipocytes induction from 3T3-L1 cells

The Western blot results showed that after FGF21 treatment, the proportion of phosphorylated Akt relative to total Akt significantly increased, indicating enhanced activation of the PI3K/Akt/mTOR signaling pathway. This demonstrated that FGF21 could improve the sensitivity of adipocytes to insulin, reflecting its favorable biological function.

Figure 1.5.3- The downstream molecules detection of FGF21 signaling using Western blot

6 The Fluorescent Localization of P9

We initially incorporated a FLAG-tag into the P9 fusion protein for subsequent identification. By utilizing antibodies that specifically bind to FLAG-tag and emit fluorescence, we conducted staining on the induced bacteria and observed afterwards. The engineered bacteria demonstrated green fluorescence emitted by the antibodies on the surface of the cells, while the wild-type bacteria performed no fluorescence, indicating that the P9 fusion protein was successfully anchored to the bacteria surface.

Figure 1.6- The fluorescence of the engineered bacteria

7 The surface-expressed P9 on engineered L. lactis strain NZ9000 stimulates the secretion of GLP-1

To test the effectiveness of the biological function of P9 (BBa_K5283019) expressed on the surface of our engineered L. lactis strain NZ9000, NCI-H716 cells were co-cultured with the engineered L. lactis strain NZ9000 for 2 h. After that, the cell supernatant was collected for GLP-1 concentration measurement, and the NCI-H716 cells were used for RT-PCR to assess the expression levels of Gcg, Pcsk1 and Pcsk2. We treated the NCI-H716 cells with four different conditions: no engineered L. lactis strain NZ9000 added, IPTG+, IPTG-, and IPTG+ with trypsin-EDTA.

Figure 1.7.1- The overall process of the experiment

By measuring the GLP-1 concentration through ELISA, we found that the GLP-1 secretion level in the IPTG+ group was significantly higher than that in the other treatment groups.

Figure 1.7.2- The ELISA result showing P9 stimulate the secretion of GLP-1

The transcription levels of Gcg, Pcsk1 and Pcsk2 were measured by RT-PCR for each group of cells. Compared to the other treatment groups, the expression of Gcg and Pcsk1 in the IPTG+ group was more pronounced.