Target module

Cycle 1: The Preliminary Construction of RGD Surface Display on Salmonella

Design

During a discussion with Professor Quan from the College of Basic Medical Sciences, Jilin University, he suggested that we could engineer bacteria to enhance their tumor-targeting capability. After reviewing the literature, we discovered that the Lpp-OmpA structure can be used to display RGD peptides on the outer membrane of Salmonella, thereby enhancing the bacteria's tumor-targeting specificity.

Build

We searched for the sequences of lpp, ompA, and RGD peptides and combined them, naming the construct Lpp-OmpA-RGD.

Test

Using Alphafold3 for structural simulation, we found that the protein structure scored an ipTM of less than 0.5, indicating that the structure is unlikely to exist stably.

Figure 1. Protein Structure Simulation of Different Types of RGD Peptides Linked to Lpp-OmpA Structure

Learn

Upon further review of the literature, we discovered that the Lpp-OmpA surface display system is actually composed of the signal peptide and the first nine residues of Braun's lipoprotein, referred to as Lpp', and five of the eight membrane-spanning segments of the OmpA porin (residues 46-159).

Cycle2: The Structural Optimization of RGD Surface Display on Salmonella

Design

After discovering the instability of the full-length OmpA structure in Cycle 1, we adjusted our design by utilizing the Lpp' and residues 46-159 of OmpA for RGD peptide display. To further validate the stability and display efficiency of this new design, we decided to assess it through molecular docking and other in silico methods.

Build

We reconstructed the correct Lpp'-OmpA(46-159)-RGD sequence.

Test

We performed modeling again using Alphafold3, and found that the structure scores (ipTM) of the newly constructed fusion protein were all above 0.5, indicating improved structural stability. However, through structure simulation alone, we could not yet assess the affinity differences.

Figure 2. Protein Structure Simulation of Different Types of RGD Peptides Linked to Lpp'-OmpA(46-159) Structure

Therefore, we decided to further investigate this issue using molecular docking. We downloaded the integrin structure file from the PDB database and performed molecular docking using PDBePISA. The results showed that Lpp'-OmpA(46-159)-RGD exhibited the best docking performance.

Figure 3. Molecular Docking Visualization of Lpp'-OmpA(46-159)-RGD with Integrin

To further validate that Lpp'-OmpA-RGD exhibited the highest affinity, we proceeded with molecular dynamics simulations. The results showed that the radius of gyration (Rg) distribution peak of Lpp'-OmpA-RGD in complex with integrin was the narrowest, indicating the highest structural rigidity post-binding, thus confirming its strongest affinity.

Figure 4. Distribution of the radius of gyration (Rg)

Learn

Before conducting wet lab experiments, it is crucial to perform dry lab predictions and simulations. Through protein structure prediction and molecular docking analysis, we can evaluate the structural stability of proteins and their binding potential with target molecules in advance. This approach not only helps optimize and adjust our designs but also increases the feasibility and success rate of the experiments. By doing so, we can minimize potential errors during the experimental phase, avoid unnecessary resource waste, and provide a solid theoretical foundation for subsequent wet lab work. This process has allowed us to deeply appreciate the significance of dry lab modeling tools in bioengineering, as they greatly enhance the efficiency and accuracy of experimental design.

Cycle 3: Validation of RGD Expression

Design

After reviewing previous iGEM parts, we found that the part BBa_K2632008 designed by iGEM18_HZAU-China includes the LPP'-OmpA(46-159)-RGD-His sequence. Given that this design closely aligns with our requirements, we decided to directly adopt this part. However, the original part used the plac promoter to regulate RGD expression, which we did not require. Therefore, we modified the part by removing the lac operator region while retaining the positive regulation by the CAP protein, effectively converting the plac promoter into a constitutive one. Additionally, the part had a His tag added to the N-terminus of RGD, which facilitated the detection of RGD expression. We decided to keep this feature in our design for ease of subsequent validation.

Build

We successfully constructed the modified sequence into a plasmid and transformed it into the Salmonella strain. Next, we prepared to validate the expression of RGD to confirm whether the modified design could effectively display RGD on the bacterial surface.

Figure 5. Schematic Representation of Lpp-OmpA-RGD

Test

Using the His tag, we detected the expression of RGD on the surface of Salmonella through flow cytometry. The results showed successful display of RGD on the bacterial surface, and the flow cytometry data demonstrated expression levels consistent with our expectations, further validating the effectiveness of our design.

Figure 6. Validation of RGD expression using flow cytometry

Learn

By referencing and modifying existing iGEM parts, we learned that utilizing established designs in genetic engineering can significantly accelerate the experimental process and help avoid redundant work. However, we also realized that adapting these designs to meet our specific needs is essential. For instance, removing unnecessary regulatory elements to achieve constitutive expression proved crucial in our case. Additionally, incorporating a His tag for protein detection greatly simplified experimental procedures, demonstrating that introducing detectable tags at the design stage can enhance both experimental efficiency and result reproducibility. This experience gave us a deeper understanding of how to make reasonable adjustments to mature designs, enabling faster and more accurate experimental validation.

Safety module

Cycle 1:Development of a Regulated Delayed Lysis System for Salmonella

Design

During a brainstorming session, our PI, Prof. Ming Yang, suggested the need to enhance the safety of Salmonella, and he provided us with the delayed lysis strain χ11802. We used this delayed lysis strain, χ11802, where the asd and murA genes were knocked out, preventing the bacteria from synthesizing an intact cell wall. These two genes were introduced into a corresponding plasmid and placed under the control of the araC pBAD promoter, ensuring that the bacteria could only survive in the presence of arabinose.

Build

We inserted the sequences for asd, murA, and araC pBAD into the plasmid. After transformation, we constructed the Salmonella strain carrying the plasmid.

Figure 1. Schematic Representation of araC pBAD-asd-murA

Test

We cultured the transformed Salmonella in test tubes and media with and without arabinose. The results showed that bacterial growth occurred only in the arabinose-containing medium, while the bacteria could not survive in the absence of arabinose.

Figure 2. Bacterial Growth in Test Tubes with LB, Arabinose, and Kanamycin

Figure 3. Bacterial Growth on LB Agar Plates with and without Arabinose

Learn

This method allowed us to precisely control the survival conditions of Salmonella and ensure that the bacteria undergo lysis and die after their therapeutic function is fulfilled. This design significantly enhances the safety of the therapy, effectively preventing bacterial persistence and potential risks post-treatment.

Cycle 2: Knockout of msbB to Reduce Bacterial Toxicity

Design

To reduce the toxicity of Salmonella and enhance its safety for use in humans, we decided to knock out the msbB gene. The msbB gene encodes a lipid A myristoyl transferase involved in the modification of lipid A in lipopolysaccharides (LPS). Knocking out msbB results in the production of structurally defective LPS, significantly reducing the endotoxin's pyrogenicity and immune stimulation.

Build

We successfully knocked out the msbB gene in Salmonella χ11802 using homologous recombination.

Test

PCR and gel electrophoresis confirmed the successful knockout of the msbB gene, and we successfully constructed the ΔmsbB χ11802 strain. This lays the groundwork for reducing strain toxicity and improving its safety.

Figure 4. BPCR Validation of msbB Gene Knockout in Salmonella

Learn

By successfully knocking out the msbB gene, we learned the effectiveness of gene editing techniques in reducing bacterial toxicity and enhancing safety. This process highlighted the potential of precise genetic modifications to regulate the pathogenicity of Salmonella, providing a safer option for its application in tumor therapy.

Cycle 3: Hypoxia-Induced Expression for Tumor-Specific Targeting

Design

To further enhance the specificity of Salmonella in targeting tumor tissues, we decided to exploit the hypoxic characteristics of the tumor microenvironment. We designed a gene expression system that is specifically activated under low oxygen conditions. We chose to use the nirB promoter, which is activated in hypoxic environments. Our plan was to use the nirB promoter to drive the expression of the hlyA gene, ensuring that hlyA would only be expressed in the low-oxygen tumor microenvironment, minimizing its effects on normal tissues.

Build

We placed the hlyA gene under the control of the nirB promoter. This plasmid was then transformed into our previously constructed Salmonella strain.

Figure 5. Schematic Representation of pnirB-hlyA

Test

The modified Salmonella was cultured under both hypoxic and normoxic conditions. We used Western blot analysis to assess the expression of the hlyA gene (encoding listeriolysin O) under these conditions. The results showed significant expression of listeriolysin O under hypoxic conditions, while its expression was either minimal or undetectable under normal oxygen levels. This confirms that the nirB promoter effectively drives hlyA expression in hypoxic environments.

Figure 6. Listeriolysin O Expression under Hypoxic Conditions

Learn

Through this experiment, we successfully validated the inducibility of the nirB promoter under hypoxic conditions, achieving tumor-specific expression of the therapeutic gene. This experiment highlighted the potential of utilizing the hypoxic nature of tumors to enhance the specificity of treatment, reducing side effects on normal tissues. We learned the importance of environment-inducible promoters in gene therapy, providing valuable insight for future designs.

Gene regulation module

Cycle 1: Design of Bacteria-Delivered shRNA Expression Plasmid

Design

Building on last year's work, we continued to use bacteria as a vector to deliver shRNA expression plasmids.

Build

We used an shRNA design tool to preliminarily construct an shRNA expression plasmid targeting the CLDN6 gene.

Learn

We learned how to properly design shRNA sequences. The key to shRNA design lies in selecting appropriate targets, avoiding off-target effects, and ensuring the stability and efficiency of the shRNA sequence. We realized that the careful selection of sequences not only enhances gene knockdown efficacy but also significantly reduces non-specific effects and uncertainties during the experiment. This experience provided us with valuable foundational knowledge for future shRNA sequence design.

Cycle 2: Development of Trans-kingdom RNAi

Design

After discussing with the BUNZH-CHINA 2023 team, we learned about their trans-kingdom RNAi (tkRNAi) system. This system enables bacteria to express shRNA, allowing the bacteria to enter tumor cells and release shRNA to induce gene silencing. Based on our communication and their experimental results, we concluded that the tkRNAi system might achieve better gene silencing compared to simply using bacteria as a vector to deliver shRNA expression plasmids. Therefore, we decided to adopt their tkRNAi approach to enhance gene silencing efficiency.

Build

We modified our shRNA expression system by incorporating BUNZH-CHINA 2023's strategy and constructed a tkRNAi plasmid.

Figure 1. Schematic Representation of tkRNAi components

Learn

Throughout this process, we learned the design principles and applications of tkRNAi. We realized the unique advantage of tkRNAi, which uses bacteria as an RNAi delivery vector, enabling cross-species shRNA transfer to regulate target genes. This approach offered a new perspective for our research, highlighting the flexibility and potential of bacteria-mediated gene delivery systems in gene regulation. The introduction of tkRNAi significantly enhanced the gene silencing effect and opened up broader potential for further applications.

Cycle 3: Improvements for tkRNAi

Design

While designing the tkRNAi plasmid, we realized that the T7 RNA Polymerase gene was too large, causing space limitations on the plasmid. To ensure that the tkRNAi system would work effectively in our study, we decided to insert the T7 RNA Polymerase gene into the genome of the previously constructed ΔmsbB χ11802 Salmonella strain. This would free up plasmid space for other critical elements.

Build

Using gene recombination techniques, we inserted the T7 RNA Polymerase gene into the Salmonella genome. See the experiment section for detailed procedures.

Figure 2. Schematic Representation of Genetic Modification of Salmonella

Test

We validated the successful integration of the T7 RNA Polymerase gene into the Salmonella genome through PCR and gel electrophoresis.

Figure 3. PCR Validation of T7 RNA Polymerase Gene Knock-in in Salmonella

Learn

By integrating the T7 RNA Polymerase gene into the Salmonella genome, we successfully constructed a new strain capable of expressing any desired gene placed under the control of the T7 promoter, enabling efficient gene expression. This modification not only expanded the functionality of our gene regulation module but also provided greater flexibility for future experiments. It allows us to express multiple genes on the same platform, significantly enhancing the system's adaptability and scalability.

Drug delivery module

Cycle 1: Design of Bacteria-Mediated Chemotherapy Drug Delivery via Nanoparticles

Design

During our initial brainstorming, Professor Ming Yang from the College of Basic Medical Sciences at Jilin University suggested that if the bacteria only carried the RNAi module, its modularity and functionality would be limited, and it would lack true innovation as an “engineered bacteria.” Since our original goal was to address the issue of chemotherapy resistance, Yang proposed that delivering chemotherapy drugs directly to the tumor via the engineered bacteria could be a valuable enhancement. This led us to the idea of using nanoparticles for drug delivery.

We explored various methods for attaching nanoparticles to bacteria, including chemical bonds, ionic bonds, or biotin-based linkages. After consulting with Professor Caina Xu from the College of Basic Medical Sciences at Jilin University, we concluded that chemical bonding would provide the highest feasibility and stability. Given the abundance of carboxyl groups on the surface of Salmonella, and referencing previous studies (we will add a citation later), we chose to use chemical bonds to link the nanoparticles to the bacteria.

Build

Based on this, we decided to modify the surface of the nanoparticles with amino groups, allowing them to form amide bonds with the carboxyl groups on the bacteria. Additionally, considering safety and cost factors, we opted to use PLGA as the drug carrier material, with PEG-NH2 serving as the linker to connect the nanoparticles to Salmonella.

Test

As we refined the nanoparticle preparation and linkage protocols, we discovered that using PEG with dual modifications might cause the nanoparticles to link to one another, resulting in reduced material efficiency.

Learn

We learned that if we could avoid using an external linker and instead treat the bacteria itself as a functional linker, we could potentially improve the material efficiency and resolve the issue of low material utilization.

Cycle 2: Redesign and Optimization of Nanoparticles for Chemotherapy Drug Delivery

Design

To address the issues identified in Cycle 1, we consulted with Professor Yuetao Zhang from the College of Chemistry at Jilin University to redesign the nanoparticles and optimize their functionality. The primary issue in Cycle 1 was that the NH2 group was located on the linker, rather than directly on the nanoparticles. If we could modify or attach NH2 directly to the nanoparticles, this problem could be resolved.

Build

Based on this new approach, we decided to use multi-block supramolecular polymers to prepare the nanoparticles. To ensure safety while having the amino groups exposed on the nanoparticle surface, we chose to utilize the amino side chains on lysine. Additionally, due to the presence of phagocytes in the bloodstream, we needed to prevent the nanoparticles from being engulfed before reaching the tumor tissue. Therefore, we decided to use mPEG to prevent phagocytosis. Considering these factors, we opted to construct nanoparticles using mPEG-PLGA-PLL triblock copolymers.

Test

To ensure professionalism and safety, our team conducted experiments under the supervision of experts from the State Key Laboratory of Supramolecular Structure and Materials at Jilin University and professionals from the chemistry department. Ultimately, we successfully obtained the mPEG-PLGA-b-PLL block copolymer.

Figure 1. mPEG-PLGA-b-PLL in DMSO-d6

Learn

We successfully synthesized the nanoparticles. To prepare for future experiments, we loaded cisplatin into the nanoparticles to validate their effectiveness in subsequent trials.