iGEM 2024 — Team lzu-medicine-china

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## April

Initial thoughts are established, and preliminary experimental design is underway.
Suitable target genes will be selected, along with appropriate chassis organisms and types of transformation plasmids.

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## May

Synthesize the gene and construct recombinant plasmids.

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## June

Prepare competent cells. Cultivate bacterial cultures and extract plasmids, followed by the transformation of the target gene into the chassis organism, Escherichia coli Nissle 1917 (ECN1917). Synthesize DLPC-encapsulated engineered bacteria.

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## July

Verification of the functions of GLP-1 and BCoAT genes includes the following steps: Protein concentration in the supernatant will be measured using the BCA protein assay kit. For the determination of secreted proteins, the supernatant will be filtered through a 0.22 µm membrane. The expression of secreted proteins will be quantified according to the instructions of the GLP-1 ELISA kit. For Western blot analysis, log-phase bacterial cultures will be collected and centrifuged at 10,000 rpm for 5 minutes. The supernatant will be collected and precipitated for colony counting. Using anti-His and anti-Flag antibodies, Western blotting will be performed to detect the negative control group, positive control group, the transformed internal reference gene protein BCoAT, and the expression of recombinant GLP-1 protein.

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## August

Assess bacterial viability using the CCK-8 assay and the DMAO/PI staining method for evaluating bacterial activity and cell death.

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## September

Utilize transmission electron microscopy to observe DLPC-encapsulated engineered bacteria.

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## Others

Our division of labor schedule.

![picture-9](https://static.igem.wiki/teams/5161/notebook/notebook.webp)

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## 1.1. First Iteration - Chassis Engineering

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### Design

Considering the need to monitor blood glucose levels and regulate glucose and lipid metabolism through the released short-chain fatty acids (SCFAs) and glucagon-like peptide-1 (GLP-1), we selected the probiotic strain Escherichia coli Nissle 1917 (ECN1917) as the chassis organism for the entire system. Additionally, since we need to test the engineered bacteria under various conditions, it is essential to assess the compatibility of the chassis organism with all experimental conditions.

### Build

To simulate different blood glucose concentrations, we designed L.B. agar plates with varying glucose levels: L.B. agar plates without glucose, L.B. agar plates with 2.0 mmol/L glucose, L.B. agar plates with 5.0 mmol/L glucose, L.B. agar plates with 8.0 mmol/L glucose, and L.B. agar plates with 11.0 mmol/L glucose, to represent low, normal, and high blood glucose ranges. Subsequently, we inoculated these agar plates with the same dilution factor of the chassis ECN1917 as well as the engineered bacteria containing the introduced plasmid to evaluate their growth status.

### Test

The results indicated that all types of bacteria grew normally on the various plates, and there were no statistically significant differences in colony counts.

### Study

Based on the above findings, we proceeded to the next iteration, which involved encapsulating the engineered bacteria within vesicles.

## 1.2. Second Iteration - Chassis Engineering

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### Design

In this iteration, we designed self-assembling nanovesicles based on 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DLPC) to prevent the potential exposure of immunogenic components and the division of engineered bacteria. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG) served as the primary component of the membrane, while cholesterol (1% w/w) was added to enhance membrane stability. Calcium ion solution was used to promote the self-assembly of the membrane on the surface of the engineered bacteria.

### Build

We diluted and inoculated both the conventional engineered bacteria and the engineered bacteria loaded in nanovesicles onto the agar plates used in the first iteration to investigate whether the nanovesicles inhibited bacterial division. Additionally, we employed DMAO/PI staining to determine whether the engineered bacteria maintained normal viability.

### Test

We found that the engineered bacteria not encapsulated in nanovesicles formed normal colonies on the plates, while those encapsulated in nanovesicles formed very few colonies. This indicates that the nanovesicles indeed inhibited bacterial division. The fluorescent staining results for the engineered bacteria demonstrated that, regardless of encapsulation in nanovesicles, the engineered bacteria exhibited viability.

### Study

Based on the aforementioned results, we proceeded to the next iteration aimed at determining whether the substances produced by the engineered bacteria could be released into the external environment of the nanovesicles.

## 1.3. Third Iteration - Chassis Engineering

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### Design

In this iteration, we validated the ability of the engineered bacteria encapsulated in nanovesicles to secrete target products, comparing them with normal engineered bacteria.

### Build

We cultured the same concentration of normal engineered bacteria and those encapsulated in nanovesicles in PBS buffer with the highest glucose concentration (11.0 mmol/L), designed to inhibit the proliferation of normal engineered bacteria due to the lack of exogenous amino acids and nucleotides. To detect the secretion of SCFAs and GLP-1 recombinant protein, we employed high-performance liquid chromatography (HPLC) and an ELISA kit for GLP-1 to measure the levels of target substances in the buffer.

### Test

The results indicated that both normal engineered bacteria and those encapsulated in nanovesicles were able to produce the target products. However, the GLP-1 yield was 53.7% of that from the normal bacteria. This suggests that although the GLP-1 recombinant protein can cross the phospholipid composite vesicles, its diffusion rate per unit time is lower.

### Study

Based on the aforementioned results, we will further investigate the expression of the target protein in the next steps.

## 2.1. First Iteration - Gene Expression Verification Engineering

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### Design

In this iteration, we need to first verify the successful transformation of the recombinant plasmid into the chassis bacteria.

### Build

Since the recombinant plasmid contains a kanamycin resistance gene, we will dilute the bacterial suspension using an electroporator and inoculate it onto kanamycin L.B. agar plates to select for resistant colonies. After that, we will pick single colonies for amplification, extract total RNA according to the requirements of the total RNA extraction kit, and perform real-time quantitative PCR using designed primers to verify whether the recombinant plasmid has been successfully introduced into the engineered bacteria.

### Test

The results indicate that the chassis bacteria without the plasmid showed no growth on the kanamycin plates, while the engineered bacteria formed colonies on the kanamycin plates. PCR results confirm that the plasmid was successfully introduced into the engineered bacteria.

### Study

Based on the above experimental results, we conducted further validation of protein expression.

## 2.2. Second Iteration - Gene Expression Verification Engineering

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### Design

First, we will verify the expression of the key protein, butyryl-CoA: acetate CoA-transferase (BCoAT), responsible for producing SCFAs in the engineered bacteria.

### Build

According to existing literature and databases, we estimate that the molecular weight of the BCoAT protein transcribed from the pET28a-BCoAT-His plasmid should be around 46 kDa. We cultured the engineered bacteria in L.B. liquid medium with the highest glucose concentration for 8 hours to reach the logarithmic growth phase, then lysed the bacteria on ice using an ultrasonic cell disruptor and centrifuged at high speed to obtain the cell-free products. Protein separation was performed using SDS-PAGE gel electrophoresis, and specific detection was carried out using His antibodies.

### Test

We have successfully isolated the BCoAT protein, and its molecular weight is consistent with the expected value.

### Study

Based on the above findings, we will further verify the expression of the second target protein.

## 2.3. Third Iteration - Gene Expression Verification Engineering

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### Design

Throughout the project, we used two strategies—enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE gel electrophoresis—to detect the expression of GLP-1 recombinant protein. In this section, we infer that the molecular weight of the GLP-1 recombinant protein is between 74-76 kDa.

### Build

We cultured the engineered bacteria in L.B. liquid medium with the highest glucose concentration for 8 hours to reach the logarithmic growth phase, then performed high-speed centrifugation to collect the supernatant. Protein concentration was determined using a BCA protein quantification kit, and proteins were separated by SDS-PAGE. Subsequently, we used FLAG tag antibodies for specific Western blot screening to capture the target protein.

### Test

The results of SDS-PAGE gel electrophoresis and Western blot indicate that the GLP-1 recombinant protein is indeed expressed in the supernatant of the engineered bacteria, confirming that it has been successfully secreted into the extracellular space.

### Study

Based on the above findings, we will conduct functional validation of the target gene in the upcoming iterations.

## 3.1. First Iteration - Gene Function Validation Engineering

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### Design

In this iteration, our primary goal is to assess the function of the BCoAT gene, specifically focusing on the rate and levels of short-chain fatty acids (SCFAs) production, primarily acetate, propionate, and butyrate.

### Build

We used high-performance liquid chromatography (HPLC) to measure the expression of three SCFAs—acetate, propionate, and butyrate—produced by engineered bacteria under time-series conditions, as an indicator of BCoAT gene functionality.

### Test

The results indicate that, compared to the chassis bacteria, the engineered bacteria were able to produce all three short-chain fatty acids (SCFAs). This confirms that the BCoAT gene is functioning normally in the engineered strains. Time-series analysis revealed that, 12 hours after inoculation into the new medium, the concentrations of the three SCFAs gradually increased, which corresponds to the transition of the engineered bacteria from the logarithmic growth phase to the stationary phase.

### Study

Although we have validated the ability of the engineered bacteria to produce SCFAs in this section, we have not confirmed the functional relationship between SCFAs and glucose regulation (although this has been reported in the literature, it does not prove that the engineered bacteria have the same effect). We plan to conduct in vitro cell experiments in the next phase to verify this effect.

## 3.2. Second Iteration - Gene Function Validation Engineering

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### Design

In this iteration, we will verify the functionality of the GLP-1 recombinant protein, primarily its role in glucose regulation through GLP-1 release.

### Build

We established a gradient of glucose concentrations in the culture medium, as well as a time series at the highest glucose concentration (simulating a hyperglycemic environment) to cultivate the engineered bacteria. Afterward, we collected the supernatant from the culture by centrifugation and used the ELISA method to measure the concentration of GLP-1 in the supernatant, inferring the results of its release.

### Test

The results indicate that, under the same cultivation time conditions, the concentration of GLP-1 in the supernatant increased with the rising glucose content in the culture medium. In the time series measurements at the highest glucose concentration (11 mmol/L), the GLP-1 concentration gradually rose over the 12-hour period and then leveled off.

### Study

Although the designed GLP-1 recombinant protein achieved the expected functionality, we did not conduct more detailed tests on the individual components of the recombinant protein, which is necessary for future work. Additionally, we need to consider whether the GLP-1 recombinant protein can exert biological value in regulating blood glucose levels, which will be validated in future cell experiments.

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## 1. Verification of the compatibility between engineered bacteria and

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### experimental conditions

To ensure that the engineered bacteria can grow under all the experimental conditions we designed (specifically in L.B. medium simulating different glucose concentrations), we diluted and spread the chassis bacteria along with the engineered bacteria containing the plasmid onto L.B. agar plates to observe the bacterial growth status.

Furthermore, in order to confirm that the experimental conditions we set up do not affect bacterial viability and protein expression, we conducted additional experiments. We used a bacterial viability assay kit (CCK-8 method) to measure bacterial viability. Note: These experiments were performed when the bacterial suspension reached an OD600 = 0.4.

### Result

We established a series of glucose concentration gradients in L.B. liquid medium and agar plates, including L.B. agar plates without glucose, L.B. agar plates with 2.0 mmol/L glucose, L.B. agar plates with 5.0 mmol/L glucose, L.B. agar plates with 8.0 mmol/L glucose, and L.B. agar plates with 11.0 mmol/L glucose.

Based on the results observed on the bacterial plates, both the chassis bacteria and the engineered bacteria grew normally on all L.B. agar plates with different glucose concentrations. The colony counts and colony morphology appeared normal without any abnormalities. This indicates that the experimental conditions we set up are reliable.

![picture-5](https://static.igem.wiki/teams/5161/proof-of-concept/image5.webp)

The results of the CCK-8 assay indicated that there were no significant differences in metabolic activity between the chassis bacteria and the engineered bacteria in L.B. medium with different glucose concentrations.

![picture-6](https://static.igem.wiki/teams/5161/proof-of-concept/image6.webp)

## 2. Detection of target gene expression

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We used the Western blot method to detect the expression of the target protein BCoAT (recombinant protein tag 5xHis) and the GLP-1 fusion protein (recombinant protein tag 3xFlag). The results indicated that BCoAT was expressed in both engineered bacteria, with a decrease in expression observed in the DLPC-encapsulated engineered bacteria. The molecular weight of BCoAT was approximately 46 kDa, consistent with the expected results. The expression trend of GLP-1 in both engineered bacteria was similar to that of BCoAT, with a molecular weight ranging between 74 to 76 kDa, also in agreement with the expected results.

![picture-7](https://static.igem.wiki/teams/5161/proof-of-concept/image7.webp)

## 3. BCoAT functional validation

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For BCoAT, we measured the concentrations of three short-chain fatty acids, including acetate, propionate, and butyrate, using high-performance liquid chromatography. The results showed a significant increase in the production of these short-chain fatty acids in DLPC-encapsulated engineered bacteria compared to the chassis bacteria (control group). However, the butyrate production in DLPC-encapsulated engineered bacteria decreased compared to normal engineered bacteria, likely due to the larger molecular weight of butyrate, which increases the resistance required to pass through the phospholipid membrane. The above experimental results were all measured at a glucose concentration of 11 mmol/L.

![picture-8](https://static.igem.wiki/teams/5161/proof-of-concept/image8.webp)

## 4. Glucose concentration-dependent gene functional validation

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The results of the glucose concentration gradient cultivation indicate that the efficiency of engineered bacteria in producing short-chain fatty acids is independent of glucose concentration, which aligns with our expected outcomes.

The concentration of the GLP-1 recombinant protein secreted by the engineered bacteria increases with higher glucose concentrations.

![picture-9](https://static.igem.wiki/teams/5161/proof-of-concept/image9.webp)
![picture-10](https://static.igem.wiki/teams/5161/proof-of-concept/image10.webp)

## 5. Time-series release of GLP-1 recombinant protein.

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The results indicate that the release of GLP-1 recombinant protein by the engineered bacteria gradually increases and stabilizes within 12 hours. The concentration of GLP-1 increases most rapidly during the bacterial logarithmic phase (6-8 hours), which is consistent with our expected outcomes.

![picture-11](https://static.igem.wiki/teams/5161/proof-of-concept/image11.webp)

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