iGEM 2024 — Team afmu-china

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %} {% block page\_content %}
![X-background](https://static.igem.wiki/teams/5283/backgroundp/notebook456.webp)

Notebook

* [February](#one)
* [March](#two)
* [April](#three)
* [May](#four)
* [June](#five)
* [July](#six)
* [August](#seven)
* [September](#eight)

# February

During this month's recruitment activities, we introduced the upcoming iGEM competition to potential new members through an online meeting. Members from the previous team shared their valuable experiences, including how to prepare for the
competition, team collaboration skills, and the impact of participating in the competition on their personal growth. This meeting not only attracted many talented students but also stimulated their interest in synthetic biology and the
iGEM competition.

# March

This month, we had the privilege of inviting Professor Han Hua to hold a lecture on synthetic biology for team members and interested students. Professor Han provided an insightful introduction to the fundamental principles of synthetic biology,
current research trends, and future application prospects. Furthermore, he encouraged students to contemplate how synthetic biology could be applied to address real-world issues, broadening their scientific perspectives and stimulating
innovative thinking among the attendees.

# April

With the addition of new members, we held a meet-and-greet this month aimed at introducing new and old members to each other, understanding the team culture, and making preliminary assignments based on each individual's interests and expertise.
This meeting provided a platform for team members to get to know each other and establish connections, laying the groundwork for subsequent project collaboration.

During our first brainstorming session of the month, team members actively proposed a variety of innovative ideas and project proposals. We engaged in in-depth discussions and corrections to narrow down these ideas, ultimately selecting several promising
proposals that laid the foundation for the next round of detailed planning and design. This brainstorming session not only stimulated the team's creativity but also strengthened communication and collaboration among members.

# May

This month, we solidified the project content for this year's iGEM competition through our second brainstorming session. Team members engaged in in-depth discussions around the chosen theme and ultimately made decision on the research direction.
Additionally, to better promote our project, we began creating posters, promotional videos, and educational comics aimed at introducing the public to the allure of synthetic biology and our project philosophy.

Concurrently, we initiated the design work for a series of lactobacillus-themed merchandise, which not only strengthens team cohesion but also serves as promotional material in subsequent events, helping us attract more attention and support.

Furthermore, we commenced work on web modeling, planning to construct a professional website to showcase our project progress and achievements, while also providing a platform for team members to communicate and collaborate.

# June

To better understand the public's awareness and attitudes towards Type 2 diabetes, we designed and distributed questionnaires this month. Through these surveys, we collected a wealth of data, providing valuable information for further research
into our project.

We successfully achieved plasmid construction and transformation. The successful construction and transformation of the plasmid is the cornerstone of our project's success, proving that our genetic design strategy is feasible and implementable.
This provides a reliable starting point for the project, ensuring that we can move forward on schedule and lays a solid foundation for subsequent experiments.

- Then we have completed experimental training, learning the operations of various experiments.

During the Dragon Boat Festival, we organized a community event to educate residents about diabetes-related knowledge, raising public health awareness.

Bacterial growth modeling was completed. Through bacterial growth modeling, we can ensure the repeatability and predictability of experiments, which is crucial for the accuracy of subsequent experiments. This step guarantees that we can test
our hypotheses in a standardized and controllable environment, providing a robust experimental platform for the project.

Additionally, we interviewed patients with Type 2 diabetes and experts in the field to gain a deeper understanding of the current state of diabetes treatment and the actual needs of patients from their perspectives, providing important references
for the project's further research.

# July

This month, we held our fourth brainstorming session and exchanged experiences with Assistant Researcher Qiu Xinyuan on our competition project. This exchange not only kept us informed about the latest developments in the iGEM competition
but also provided us with valuable strategies and suggestions for participation.

We completed the experiment of inducing bacteria to produce FGF21 under stress. Through this experiment, we verified that our project's engineered bacteria can serve as an effective bioreactor for producing proteins related to diabetes treatment.
This not only proves the innovativeness of our approach but also offers a potential new avenue for diabetes therapy.

We further completed the purification and Western Blot (WB) verification of FGF21, further confirming the functionality of our engineered bacterial system and the biological activity of the produced protein. This provides significant evidence
for the success of our project, indicating that we can produce bio-molecules with therapeutic potential for use in future treatment strategies.

![notebook-2](https://static.igem.wiki/teams/5283/notebook-img/9.webp)

Concurrently, we engaged in in-depth exchanges with other GEM teams at the 11th CCiC conference, sharing our project philosophy and progress while also learning from the excellent experiences of other teams.

Additionally, we interviewed Teacher Zhang Qiuju from the Ethics Teaching and Research Office to discuss biosecurity ethics and the impact of our project on humans and society, ensuring that our project meets ethical and social responsibility
requirements while exploring science.

To better showcase our project outcomes, we reported on the progress of our website design and arranged work for the art team, strengthening communication and collaboration among team members to ensure that the website accurately and aesthetically
presents our project content.

# August

We participated in the 6th Northwest Exchange Conference, sharing our research findings with other teams and learning about outstanding projects from them. This conference not only strengthened our connections with other teams but also provided
us with valuable feedback and suggestions. And this is a part of our experimental documentation.

We also designed and completed an experiment to improve insulin resistance in adipocytes with FGF21, demonstrating that FGF21 can significantly enhance insulin sensitivity in 3T3-L1 differentiated adipocytes, offering new hope for cellular
therapy in diabetes. This discovery confirms the potential of our engineered bacterial strategy in regulating cellular metabolism and improving the pathological state of diabetes.

We successfully engaged in an exchange with the HP team from Hainan University. During this event, we shared our project experiences, discussed the latest advancements in the field of synthetic biology, and learned valuable knowledge and experience
from their work. This exchange greatly broadened our horizons and strengthened our ties with the HP team from Hainan University.

# September

We completed the P9 membrane surface fluorescence localization experiment, and the successful anchoring of the P9 protein on the bacterial surface demonstrated our ability to precisely control the bacterial surface display system. This provided
a key technical validation for the project and offered an effective platform for subsequent research on the function of the P9 protein and its application in diabetes treatment.

We completed the experiment of P9 stimulation of GLP-1 secretion in NCI-H716 cells, and by verifying the stimulation of GLP-1 secretion by the P9 protein, we confirmed the potential of our engineered bacterial strategy in regulating the secretion
of intestinal hormones. This result provided an important molecular mechanism for the project, aiding us in further exploring and optimizing diabetes treatment strategies.

We further confirmed through RT-PCR that the stimulation of GLP-1 secretion by the P9 protein and the upregulation of related pathway genes solidified the role of engineered bacteria in regulating the secretion of intestinal hormones, and
provided a key molecular mechanism for the development of diabetes treatment strategies.

Our team completed the production of a demonstration video, vividly showcasing our innovative strategy and project progress in treating Type 2 diabetes using engineered lactobacillus.

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement
should be distinct from your Contribution for Bronze.{% endblock %} {% block page\_content %}
![X-background](https://static.igem.wiki/teams/5283/notebook-img/wetlab-background.webp)

Engineering

* [Overview](#one)
* [Protein Expression and Secretion](#two)
* [Molecular Structure](#three)

# Overview

##### “Failure is simply the opportunity to begin again, this time more intelligently. ——Henry Ford”

Despite in car engineering or biological engineering, the Design-Build-Test-Learn cycle is a crucial methodology in engineering that emphasizes iterative improvement just as Mr. Ford summarized. By designing a prototype, building it, testing
its performance, and learning from the results, whether negative or postive, we can systematically identify flaws and areas for enhancement. This iterative process allows our team to make informed decisions based on empirical data rather
than assumptions. Each cycle not only refines the current design but also fosters innovation by encouraging experimentation and adaptation. Ultimately, this approach leads to more effective, reliable, and more comprehensive solutions,
significantly improving the overall quality of the engineering of our project.

#### Have they used models to meaningfully predict the behavior of their system or guide their experimental or design choices, or alternatively, have they subsequently built models that characterize and explain how their system works?

In early stage, we had brainstorms and discussions about the preliminary designing and as we progress, we had several meet-ups to conduct experiments and discuss about the results, and get inspired from failures to come up with better ideas.
Throughout our project, we fully adhere the iterative “design-build-test-learn” cycle of engineering.

#### What experiments did the team do, and were the data replicated or built upon?

We conducted various experiments throughout cycles, which include using bile acid to stimulate our expression system we successfully produced our target molecules and we extracted and purified them afterwards for further cell experiments.

#### How rigorous are their experimental designs and measurements?

We represent our extremely rigorous designing in details at our [result page](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%7D%7D#one) and [protocol page](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%7D%7D#one). We strictly follow the safety guidelines and standardized procedures during our experiments. We also took a variety of measurements for
our results to reach the best criteria and the most persuasive conclusion. Details of our measurements can be found in the [result page](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%7D%7D#one).

#### How much attention have the teams given to making the progress they have made reusable? For parts, or part collections, how well characterized are they? Is this clearly documented in the Registry? Would you be happy to use these parts?

We have standardized and uploaded the parts we used in our project to meet the iGEM community requirements. We welcome and appreciate you to use our part and please do contact us if you have any enquiries about its usage. You can learn more
in the [parts page](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27parts%27%29%7D%7D#one).

# Project design

## Protein Expression and Secretion

### Cycle 1- Food-borne Stress Induced Expression of Both Molecules

#### Design

Currently, a variety of treatments for type 2 diabetes are available and continuously evolving. However, each approach have some inherent limitations, whether being invasive or not sufficiently durable. Traditional therapies, such as pharmacological
interventions, may have a range of adverse effects and impose significant physical and psychological stress on patients due to frequent insulin injections. Conversely, more innovative treatments, like insulin pumps, also rely on invasive
implantation procedures, which may not be ideal for all patients.

Therefore, after some literature searching and reading, we found **Fibroblast Growth Factor 21(FGF21)** and **P9 protein** as ideal molecules that we can express with synthetic biology methods.

FGF21 is a member of the fibroblast growth factor family, which plays a crucial role in various physiological processes, including metabolism, insulin sensitivity, and energy homeostasis. Insulin resistence of patients is
a pain point in t2dm treatment, so we chose FGF21 as a molecule to express and secrete from our engineered bacteria. Lactobacillus lactis is itself a part of the intestinal flora, so it can minimize some potential
safety concerns, its also widely used as an engineering bacteria which offered us more designing options.

The P9 protein derived from Akkermansia muciniphila, has garnered significant attention due to its unique properties and potential health benefits. This protein is involved in various metabolic
processes and plays a crucial role in maintaining gut homeostasis. Studies have shown that it can stimulate GLP-1 seceretion of human intestine cells, which can contribute to the trending GLP-1 therapy for T2DM.

#### Build

A practical challenge is controlling the expression of FGF21 and P9, as persistent secretion of these factors may lead to potential side effects, which we seek to avoid. Therefore, we aim to regulate the expression of these molecules in accordance
with the postprandial blood glucose variation curve. This approach is particularly crucial, as postprandial periods represent a critical challenge for diabetes patients. By targeting expression during these times, we hope to address the
most difficult phase of glycemic control.

The GroESL promoter was chosen as the key module to realize postprandial secretion of both molecules. It can function under **bile acid stress stimulation** after meals and start the expression of FGF21 and P9.

![eng-1](https://static.igem.wiki/teams/5283/eng-img/eng-1.webp)

###### Figure 1- The designed sequence for induced expression of FGF21.

#### Test

We used software to simulate the binding of FGF21 with its receptor, details can be seen in the molecular simulation iteration.

We initially followed the conditions from a reference, but the results shown that the absence of target molecules in the supernatant is inconsistent with the findings reported in the reference.

![eng-2-1](https://static.igem.wiki/teams/5283/eng-img/eng-2-1.webp)
![eng-2-2](https://static.igem.wiki/teams/5283/eng-img/eng-2-2.webp)

###### Figure 2- The ELISA result of FGF21 under different stimulation condition.

#### Learn

To address this issue, we consulted our advisor and senior researchers in the laboratory who have conducted similar studies. We concluded that the negative results may be attributed to the short stimulation time, which likely prevented the
expression system from completing a full cycle of expression and secretion. So we changed our stimulation condition and more details can be seen in [results](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%7D%7D#one) page. We also considered that under conditions of induced expression for both
molecules, their secretion efficiency would be impacted, potentially hindering their optimal efficacy.

### Cycle 2- Constitutive Expression of P9

#### Design

To avoid the reduction in efficiency caused by the competition between the two secretion of the two molecules, we decided to employ constitutive expression for P9. Constitutive expression utilizes fewer resources and allows for continuous
expression over an extended period, thereby ensuring an adequate supply of receptors and maximizing the functional effect.

#### Build

To better present P9, we designed the P32 promoter , the P9 sequence, and an cA anchoring protein sequence together. By employing this approach, we aim to anchor P9 **on the bacteria surface**, thereby facilitating
direct stimulation of GLP-1 secretion and enhancing its therapeutic efficacy.

![eng-2](https://static.igem.wiki/teams/5283/eng-img/eng-2.webp)

###### Figure 3- The designed sequence for constitutive expression of P9.

#### Test

We also employed software to simulate the structure of the P9 fusion protein, and the results indicated that it did not impact the functionality of P9 itself, more details can be seen in the molecular structure iteration.

Additionally, we performed immunofluorescence staining using a fluorescent antibody specific to the FLAG tag, which demonstrated that the fusion protein was presented on the bacterial surface. More details about the experiment can be seen in [result](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%7D%7D#one)

![eng-3](https://static.igem.wiki/teams/5283/eng-img/eng-3-1.webp)

###### Figure 4- The fluorescence of engineered bacteria.

#### Learn

During the iterative process, we realized the importance of not confining our efforts to theoretical considerations alone; it is equally essential to take practical issues such as expression efficiency into account. Moreover, after encountering
failures in reproducibility, we recognized the necessity of actively adjusting our experimental approach in order to successfully obtain results.

## Molecular Structure

### Cycle 1- The FGF21 Fusion Protein

#### Design

Through some literature reading, we have observed that the passage of FGF21 through the bacteria membrane is relatively challenging. Therefore, we aim to facilitate this process by employing a fusion expression approach, combining FGF21 with
an auxiliary factor to enhance its secretion.

#### Build

We chose LMWP to form the fusion protein with FGF21. It can enhance the membrane translocation capability of FGF21. We also identified the receptor structure of FGF21 from literature and performed simulations of our fusion
protein and its interaction with the receptor.

#### Test

We chose to combine LMWP with the **C-terminus** of FGF21 and conducted structural modeling.

![eng-4](https://static.igem.wiki/teams/5283/eng-img/eng-4.webp)

###### Figure 5- The structure of the first FGF21 fusion protein.

And simulated its combination with the receptor afterwards.

![eng-5](https://static.igem.wiki/teams/5283/eng-img/eng-5.webp)

###### Figure 6- The combination simulation of FGF21 and its receptor.

#### Learn

Upon investigation, we discovered that the fibroblast growth factor receptor (FGFR) exists as a dimer of approximately 58 kDa within the cell, with a distance that can reach up to 105 angstroms, which is significantly greater than the 7.3
angstroms separation between LMWP and the active site of FGF21. Consequently, we have confirmed that the first generation of FGF21-LMWP fusion protein was unsuccessful.

### Cycle 2- Improvement of FGF21 Fusion Protein

#### Design

Given that our previous design obstructed the active binding site of FGF21 with its receptor, we need to redesign the structure of the fusion protein to facilitate effective binding with its receptor.

#### Build

Due to the previous binding of LMWP to the C-terminus of FGF21 obstructing the active binding site for the interaction of FGF21 with its receptor, we have chosen to recombine LMWP with the **N-terminus** of FGF21 in hopes of exposing
the active site of FGF21.

#### Test

Using PyMOL for visualization, we examined the three-dimensional conformation of the FGF21-LMWP complex. As shown in the picture, the active sites of FGF21 are highlighted in magenta, while the LMWP segment is depicted in yellow. The LMWP
peptide displays a relatively loose conformation with an undefined spatial structure and low polarity. These characteristics suggest minimal risk of detrimental intramolecular interactions between LMWP and FGF21, which could otherwise
obstruct FGF21's binding sites to its receptor, FGFR.

![eng-6](https://static.igem.wiki/teams/5283/eng-img/eng-6.webp)

###### Figure 7- The structure of the second FGF21 fusion protein.

#### Learn

Through software simulations, we gained a comprehensive understanding of the guiding role of structural prediction in experimental design. Similarly, we conducted relevant structural simulations for the P9 fusion protein.

### Cycle 3- The P9 Fusion Protein

#### Design

Inspired by the structural simulations of the FGF21 fusion protein, we also aim to validate the feasibility of the P9 and anchoring protein fusion protein through software structural simulations.

#### Build

As previously mentioned, we aim to achieve the fusion expression of P9 with the anchored protein to enable its immobilization on the surface of the bacterial cell. Therefore, we continued to predict its structure to assess whether this modification
would impact the normal function of the P9 protein.

#### Test

Regarding the P9 protein, there is currently no research on its active sites, so we used AlphaFold2 for molecular docking. In the figure, the green portion represents the P9 protein, the yellow represents the P9 protein receptor ICAM2, and
the light blue is the cA anchoring protein.

After finding P9's active site, considering of our previous experiences in the simulation of FGF21 fusion protein, we tried to attach the anchored protein to the N-terminus of the P9 protein and simulated its structure. And the hydropathy
predictions indicate that the P9 protein and the cA anchor protein have **strong hydrophobic interactions**, which facilitates their tight binding in aqueous solutions rather than forming a loose structure that might potentially
obscure active sites, more details can be seen in [modeling](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27model%27%29%7D%7D#one)

![eng-7](https://static.igem.wiki/teams/5283/eng-img/eng-7.webp)

###### Figure 8- The structure of the first P9 fusion protein.

![eng-8](https://static.igem.wiki/teams/5283/eng-img/eng-8.webp)

###### Figure 9- The combination of P9 fusion protein and its receptor.

#### Learn

Through structural simulations of the two fusion proteins, we mitigated potential factors that could lead to experimental failure and provided a better molecular understanding of the experimental principles. This not only contributes to the
modeling aspects of our project but also represents an indispensable component in our engineering success.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead %}You can describe the results of your project and your future plans here.{% endblock %} {% block page\_content %}
![X-background](https://static.igem.wiki/teams/5283/backgroundp/result1.webp)

Results

* [1 Successfully construct two plasmids including FGF21 and P9](#one)
* [2 Set up optimal condition for engineering bacteria growth](#two)
* [3 Successfully induce FGF21 expression](#three)
* [4 Successfully purify FGF21 protein](#four)
* [5 FGF21 secreted by engineered L. lactis improves insulin resistance](#five)
* [6 The localization of P9 on bacterial surface by immunofluorescent staining](#six)
* [7 The surface-expressed P9 on engineered L. lactis stimulates the secretion of GLP-1.](#seven)

# 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.

![result-1](https://static.igem.wiki/teams/5283/result-img/br-1-3.webp)

###### Figure 1.1.1- The construction of FGF21 plasmid

![result-1](https://static.igem.wiki/teams/5283/result-img/br-1-4.webp)

###### Figure 1.1.2- The construction of P9 plasmid

![result-1](https://static.igem.wiki/teams/5283/result-img/br-1-3-2.webp)

###### Figure 1.1.3- The sequencing result of FGF21 plasmid

![result-1](https://static.igem.wiki/teams/5283/result-img/br-1-3-1.webp)

###### 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.

![result-2](https://static.igem.wiki/teams/5283/result-img/1.webp)
![result-2](https://static.igem.wiki/teams/5283/result-img/result-1.webp)

###### 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.

![result-3](https://static.igem.wiki/teams/5283/design-img/dsgn-3-11new.webp)
![result-3](https://static.igem.wiki/teams/5283/result-img/br-2-2.webp)

###### 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.

![result-3](https://static.igem.wiki/teams/5283/design-img/dsgn-3-10new.webp)
![result-3](https://static.igem.wiki/teams/5283/result-img/br-2-4.webp)

###### 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.

![result-4](https://static.igem.wiki/teams/5283/result-img/br-3-1.webp)

###### 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.

![result-5](https://static.igem.wiki/teams/5283/result-img/result-5-1new2.webp)

###### Figure 1.5.1- The overall process of verification

![result-5](https://static.igem.wiki/teams/5283/result-img/result-5-2.webp)

###### 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.

![result-5](https://static.igem.wiki/teams/5283/result-img/result-5-3.webp)

###### 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.

![result-6](https://static.igem.wiki/teams/5283/result-img/res-1-6.webp)

###### 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.

![result-7](https://static.igem.wiki/teams/5283/result-img/result-8-1new2.webp)

###### 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.

![result-7](https://static.igem.wiki/teams/5283/result-img/result-8-2.webp)

###### 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.

![result-7](https://static.igem.wiki/teams/5283/result-img/result-8-3.webp)

###### Figure 1.7.3- The qRT-PCR result of GLP-1gene expression

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