In Vivo Experiments With Engineered Bacteria
Based on the above experimental results, we ultimately selected Mel as the anti-inflammatory factor and synthesized the target plasmid with adhesion factor (CMC) and reactive oxygen species promoter (P [OSR]). The gene was cloned in pET-28a(+) by BamHI/XhoI. (Figure1) The target plasmid was transformed into Escherichia coli strain Nissle 1917 (EcN) and the engineered strain FMK was synthesized, which was named iEraser by our team. Next, we will verify the anti-inflammatory effect of the synthesized strain in mice.
Figure 1
Order a batch of female Balb/cJNju-Foxn1nu/Nju Hfh11nu mice and conduct experiments according to the procedures shown in the table. All liquids were administered into the mice by gavage, and the concentrations of Salmonella and FMK were resuspended in PBS at a concentration of . The intestinal specimens taken were from the cecum to the colon.
The colon length of each group of mice was measured, and the results showed that the engineered bacteria could significantly alleviate the shortened colon length caused by intestinal inflammation in mice. (Figure 2A)
Figure 2A
Take some colon tissue for microscopic observation. After dehydration, embedding, and sectioning of colon tissue, HE staining was performed, and the results are shown in Figure 3A. Compared with the PBS group, the S.Tm group showed damage to intestinal epithelial cells and disrupted intestinal mucosal integrity, while the symptoms of colitis in the mouse model treated with FMK were significantly alleviated. Some tissue sections were processed for immunofluorescence staining as shown in Figure 3B. It can be seen that the IL-6 fluorescence signal intensity of tissues treated with Salmonella by gavage was significantly higher than that of PBS group, while the IL-6 fluorescence intensity of tissues treated with engineered bacteria FMK was significantly lower than that of the S group, indicating that the synthetic bacteria FMK has a significant anti-inflammatory effect.
Figure 3A
Figure 3B
Take a portion of colon tissue, completely dissociate it, stain immune factors and immune cell marker proteins, and then use flow cytometry to measure their expression levels. As shown in Figure 4A, after oral administration of FMK, the levels of IL-6, IFN-γ, F4/80, and Lys6G in the colon tissue of mice with enteritis significantly decreased. Due to the fact that F4/80 and Lys6G are the marker proteins of immune cells, the decrease in their levels indicates that the synthetic strain FMK can significantly reduce the aggregation of immune cells near colitis tissue, thereby alleviating colitis in mice.
After extracting RNA from some colon tissue cells, RT-qPCR was performed to detect the expression levels of various inflammatory factors in the cells. The results are shown in Figure 4B. Compared with the control group, the levels of pro-inflammatory factors IL-6, IL-1 β, and IL-8 in the intestinal tissue treated with FMK were significantly reduced, while the levels of anti-inflammatory factors TGF-β and IL-10 were significantly increased.
Figure 4A
Figure 4B
In order to further investigate the effects of the engineered bacterium FMK on the gut microbiota of IBD mice, we conducted the following series of treatments.
Take colon tissue from mice, extract total DNA from the sample, perform PCR amplification, purify, quantify, and homogenize the product to form a sequencing library. The constructed library is first subjected to library quality inspection and sequencing, and the results are converted into the original sequence through base calling analysis. Next, a series of operations such as filtering the raw data, identifying and removing primer sequences, denoising, and concatenating double ended sequences are performed to obtain the final valid data.
ASVs (amplicon sequence variants) represent real biological sequences that can distinguish differences between sequences with precision in single nucleotides, enabling the identification of real species in samples. Use the dada2 method in QIIME2 2020.6 software to denoise the sequence and obtain ASVs, determining the presence of species in the sample.
Perform inter group ANOVA analysis at the Phylum level, as shown in Figure 5A. At the phylum level, the engineered bacterium FMK significantly increased the abundance of Firmicutes and Verrucomicrobota in the mouse gut, while reducing the abundance of Proteobacteria and Bacteroidota (Figure 5A). Previous studies have shown a decrease in the number of Firmicutes and an enrichment of Proteobacteria in the intestinal tract of IBD. However, this phenomenon was improved after the use of FMK. Therefore, FMK has a significant function in improving the structure of intestinal microbiota, promoting the enrichment of beneficial bacteria, and reducing the number of harmful bacteria. The heatmap analysis of sample species composition and relative abundance, as well as the sample community distribution map of species evolutionary tree, are shown in Figures 5B and 5C.
Figure 5A Bar chart of intergroup analysis of variance
Figure 5B Heat map of taxonomic relative abundance clustering
Perform species heatmap analysis based on the species composition and relative abundance of each sample, extract species at the phylum level, use R language tools for mapping, and perform heatmap clustering analysis. The values corresponding to the heatmap in the figure represent the Zscore normalization of the same species across different samples using the R scale function. The color gradient from blue to red indicates the abundance from low to high among samples, and the colors correspond to the column in the graph.
Figure 5C Sample community distribution map of Phylogenetic tree
Select the feature sequences with the top 80 abundance ratios at the OTU/ASV taxonomic level, perform multiple sequence alignment using the QIIME command line, and construct a phylogenetic tree. Then, combine the phylogenetic tree and species taxonomic abundance data, and visualize them using the R package ggtreeExtra in the form of a species circular phylogenetic tree and bar chart.
Figure 6A shows the Shannon index rarefaction curve plotted using Mothur software and R language tool based on the sequencing volume of each sample at different sequencing depths. It can be seen that when the curve tends to flatten, except for the BCK1 group, the Shannon index in the group using the engineered bacterium FMK is higher than that in the IBD mouse group, indicating that FMK has the effect of enriching intestinal microbial diversity. Similar conclusions can also be drawn based on the rank-abundance curve(Figure 6B).
To describe the beta diversity of the two sets of data, we plotted the principal component analysis (PCA) of the samples, and the results are shown in Figure 6C.
Figure 6A Shannon index rarefaction curve
Figure 6B Rank Abundance Curve
Figure 6C PCA analysis chart
Overall, administering the engineered bacteria FMK designed by us to a mouse colitis model by gavage resulted in a recovery of colon length shortening, inhibition of immune cell recruitment, enrichment of beneficial bacteria in the gut, an increase in gut microbiota diversity. The upregulation of pro-inflammatory factors and the downregulation of anti-inflammatory factors are also inhibited accordingly. The above results indicate that the engineered bacterium FMK synthesized by our team can effectively alleviate the symptoms of enteritis in model mice.
The Journey of Hope for Engineered Bacteria
In this era of human struggle against diseases, Inflammatory Bowel Disease (IBD) has become a significant challenge for many patients. Countless individuals feel helpless in their battle with this condition, as the limitations of traditional treatments erode their hope for recovery. Against this backdrop, our project emerged, aiming to bring new hope to IBD patients through the innovative design of engineered bacteria.
Our project utilizes engineered Escherichia coli as a vector, designing plasmids capable of spatiotemporal expression of anti-inflammatory factors. This innovative approach not only overcomes the limitations of traditional drug therapies but also offers patients new treatment options. We employ synthetic biology techniques, supported by mathematical modeling, to continuously explore IBD treatment methods within the design-build-test-learn cycle.
To design the anti-inflammatory component, we conducted extensive research and screening, ultimately selecting Mel and Indo for our studies. We artificially melittin by connecting two bee venom peptide monomer molecules together through a linker to form a unique hairpin structure. This modification has brought about performance improvements in many aspects. We established cell and mouse inflammation models using Salmonella infection to validate and compare the effectiveness of these anti-inflammatory peptides across various dimensions. Our findings revealed that Mel demonstrated superior anti-inflammatory performance compared to Indo across all measured indicators, leading us to choose Mel for further experimental research.
At the same time, in order to ensure that our engineered bacteria only work at the right time and place, we thought that the high ROS environment in the intestines of patients with enteritis could serve as a switch for gene expression., particularly by utilizing the ROS-responsive OxyR regulatory factor. OxyR regulates multiple genes, including katG, dps, and gorA. Based on experimental results, the promoter of the katG gene was deemed suitable for the design of engineered bacteria.
The complex intestinal environment presents a significant challenge for the stable survival of engineered bacteria. To address this, we enhanced bacterial adhesion by incorporating adhesive factors, allowing the engineered bacteria to attach more effectively to the intestinal wall. This prolongs their presence in the body and ensures the sustained release of anti-inflammatory factors. Notably, the project of adhesion protein is our laboratory design a new protein：we use one of the main components of intestinal mucus glycoprotein and probiotics usually exposed the characteristics of glucan, hope to design glucan binding domain to realize the intestinal adhesion, at the same time it can also combine probiotics, regulate the intestinal microbial environment. This idea inspired us to design the CBMcipc domain, which is derived from the C. cellolytic scaffold protein CipC. To ensure optimal performance, we ingeniously incorporated additional protein sequences during the design process, creating various binding proteins. Through a series of rigorous experiments and inflammation tests, we identified the CMC protein with the highest adhesion rate and optimal probiotic binding ratio. This choice not only enhances the overall effectiveness of the project but also lays a solid foundation for future research and applications.
Finally, we cloned the anti-inflammatory factor (Mel), adhesive factor (CMC), and ROS-responsive promoter (P[OSR]) into the plasmid pET-28a(+). The synthesized target plasmid was transformed into Escherichia coli EcN, creating the engineered strain FMK, which we named iEraser. Our experiments further validated the excellent anti-inflammatory effects of the engineered bacteria FMK (iEraser) in mice, successfully alleviating intestinal inflammation. This discovery demonstrates the feasibility of FMK as a potential therapeutic approach, laying a solid foundation for developing new treatments for Inflammatory Bowel Disease (IBD) and showing promising clinical application potential. The successful construction and validation of the engineered bacteria FMK provide crucial support for the development of innovative therapies. This targeted approach enhances treatment efficacy, rapidly alleviating patients' inflammatory symptoms.
However, the progress of the experiment hasn't always been smooth. As young project members, we are facing significant challenges posed by complex experiments. In the first two months, the atmosphere in the lab was somewhat heavy. Cell cultures repeatedly failed, with cells either dying prematurely or experimental data falling short of expectations. The language problem also creates great resistance to handling official information from iGEM and using official tools. Every day, we worked hard to identify the root causes of these issues, always feeling just a step away from success.
Despite these setbacks, we remained undeterred. We held multiple meetings to scrutinize each step, actively reflecting and summarizing our experiences. We conducted daily reviews, meticulously recording every detail of the experiments to identify potential improvements. At the same time, we sought external assistance, reaching out to professors and experts from other labs for advice and learning new techniques.Gradually, we adjusted experimental conditions and optimized techniques and methods. Through continuous trial and error, the experiments finally showed progress. Seeing the healthy growth of cells in the culture dishes brought long-awaited smiles to our faces. It was this persistence and effort that put our project back on track.
What we gained from the project was not only technical advancement but also the spirit of teamwork, resilience, and the relentless pursuit of excellence. These qualities enable us to continue moving forward with confidence in the face of challenges.
At the initial stage of the project, we engaged in extensive discussions about the experimental subjects and methods, ultimately leading to the engineering bacteria experiments of this project. Due to time constraints, some steps in our experiments remain incomplete, such as the validation of adhesion factors and the limited time points in microbial diversity experiments. Additionally, due to the design scale, our project components are relatively few, currently focusing on polypeptides and simple proteins. To address this, our team conducted a brainstorming session to introduce more interesting mechanisms into the project, aiming to enhance the experimental design. These include miRNA components, SCFAs, and DL-endopeptidase; please refer to the Idea Library for details. In the future, we hope to continuously improve the project to address these problems and create more novel components. In terms of experiments, we did not achieve perfection. For example, in the microbial diversity experiment, we are doing a more complete and formal supplementary experiment to make the experiment more rigorous and designed. This time, our experimental group will include engineered bacteria containing only adhesion factors and engineered bacteria containing only anti-inflammatory factors to further verify their respective mechanisms of action. In addition, the modules of the safety plasmid have not been experimentally verified, and we hope that future teams can get inspiration from them and complete more rigorous testing.
IBD patients often experience a significant decline in quality of life. Our project aims to help them regain normalcy and hope for the future through effective treatments. Behind this project is our team's deep care and hope for every IBD patient. We believe that through continuous exploration and effort, engineered bacteria can provide not only physiological improvements but also emotional comfort. In the future, we hope to extend this innovative treatment to a broader patient population, allowing more people to benefit from scientific advancements and human compassion.