• Original innovation is important across all sciences but is especially important in synthetic biology. Novel designs and projects require a comprehensive understanding of underlying principles to weave together ideas and bring a project to life. Whether considering the physiological ramifications of a signaling cascade or transforming bacteria, synthetic biology covers a wide breadth of expertise, and each aspect is important to consider when constructing new systems.
• Similar to how established engineering disciplines such as aerospace engineering have evolved, we too are advancing towards greater predictability and control in biology. As participants in iGEM, we are thrilled to be at the forefront of this endeavor, contributing to the evolution of tools and methodologies in biological engineering. With determination and commitment, we strive to exemplify engineering brilliance and further extend the limits of synthetic biology's potential.
• The T-SAT project aims to develop a novel therapeutic strategy utilizing engineered Salmonella strains to target and induce apoptosis in cancer cells. Critical components of this system include the recognition system, the delivery system, and the killing system coupled with a bio-switch. The recognition system contains a Human Epidermal growth factor Receptor 2 (HER2) single-chain variable fragment (scFv) （BBa_K5363017) expressed by a Salmonella surface display system. The delivery system, which can induce Salmonella's self-lysis after Salmonella invades human cells, uses a PsseJ-lyseE gene circuit. Lastly, the killing system consists of an effector gene to induce cancer cell apoptosis and a synthetic translation initiation factor (STIF)-based bio-switch that ensures the expression of effector genes is confined exclusively to cancer cells. This section details the engineering efforts undertaken to verify the functionality and specificity of our project.

• We set out to create a new therapeutic for treating solid tumors. Solid tumors are notoriously hard to treat owing to several properties. They are typically immunosuppressive, necrotic, nonvascularized, and hypoxic. All of these features block traditional treatments. However, the necrotic, hypoxic, and immunologically under-surveilled core is a perfect home for many bacteria. Taking advantage of this, we decided to use Salmonella typhimurium strain VNP20009. This strain was modified not to produce LPS or purines, making it much less immunogenic and pathogenic. (Clairmont et al., 2000)
• In our first iteration, we aimed to add a single-chain variable fragment (scFv) to a Salmonella expression system to recognize tumor-specific antigens. We found a fusion protein of a human epidermal growth factor receptor 2 (HER2) scFv(BBa_K5363017) with the Salmonella Lpp-OmpA expression system. We also sought to add a fluorescence marker to visualize the bacterial colonization of tumors. To supplement bacterial colonization, we also looked for a signal transduction system with the scFv, much like the CAR-T system, to add a customizable response. (Francisco et al., 1992; Jeiranikhameneh et al., 2017)

• We started by designing the recognition system in E.coli. We use the Lpp-OmpA surface display system to express the HER2-scFv(BBa_K5363017) on the surface of E.coli.

Lpp-OmpA-HER2_scFv Plasmid Construction
• To better demonstrate the population distribution of Salmonella, we utilize the LuxI-LuxR(BBa_K5363021) circuit to express the superfolder green fluorescent protein (sfGFP)(BBa_K5363023). We aim to visualize Salmonella by introducing a plasmid named pTD103(BBa_K5363031). The GFP gene within the plasmid enables the modified bacteria to emit green fluorescence. The plasmid also contains a two-component system: LuxI-LuxR(BBa_K5363021), which is controlled by the promoter pLuxI(BBa_K5363018). This promoter induces the expression of an autoinducer, AHL. AHL binds to LuxR(BBa_K5363021) and transcriptionally activates the promoter, thereby inducing the expression of GFP. AHL can diffuse to neighboring cells, providing a mechanism for intercellular synchronization. Consequently, the concentration of AHL reflects the size of the bacterial population, and the intensity of green fluorescence can, to some extent, indicate the bacterial population density.

LuxI-LuxR-GFP Circuit Construction

• We first tested the recognition and reporter system in E. coli. We expressed HER2 scFv(BBa_K5363017) separately without a surface display system to get its recognition efficacy to HER2 antigen on human cells. Then we confirmed the fluorescence of the luxI-sfGFP(BBa_K5363031) transformed E. coli compared to that of E. coli that was transformed with sfGFP(BBa_K5363023) expressed under pBAD promoter without arabinose.

• With confirmation that the Lpp-OmpA-HER2_scFv expression system was successful, we decided to push forward the Salmonella design. Improving on this initial design, we looked for a method to kill cancer cells upon invasion by bacteria. We also reviewed the literature for bacterial signal transduction systems that might streamline the recognition of cancer cells; however, no clear options were available. Given our current system has increased recognition of cancer cells, we also read literature for ways to improve bacterial invasion into cancer cells.

• We were already aware of Salmonella's strong invasive action toward epithelial cells. Our next step was to determine how to utilize Salmonella to introduce plasmids into cancer cells and identify the specific plasmids for introduction. After discussions with Dr. Yanbo MAO, we devised a creative method involving two types of Salmonella: Helper Salmonella (HS) and Killer Salmonella (KS).
• Killer Salmonella is engineered with an scFv recognition system and a killing system plasmid to target and eliminate cancer cells. However, it lacks the ability to invade cells due to a deletion of the flagellum gene. In contrast, Helper Salmonella retains its invasive capability.
• During the treatment process, Killer Salmonella is first injected into the tumor, where it binds to the surface of target cancer cells. Subsequently, Helper Salmonella is injected and invades cells randomly. Killer Salmonella then follows Helper Salmonella into the target cancer cells, disrupting the endosomal compartment and directly releasing the plasmid containing the killer gene.
• Regarding the killing system, our objective was to induce programmed cell death, thereby reducing the risk of cytokine storms and other toxicities associated with current cancer treatments. Extensive research has demonstrated that the BAX gene (Bcl-2 Associated X-protein)(BBa_K5363000), a pro-apoptotic member of the Bcl-2 gene family, can effectively induce apoptosis in various types of cancer (Liu et al., 2016). Therefore, we constructed a plasmid containing the BAX gene(BBa_K5363000) under the control of a constitutive CMV promoter.

• Firstly, to delete the flagellum gene in Salmonella enterica serovar Typhimurium strain VNP20009 of KS, we employed the CRISPR-Cas9 method to specifically delete the flhD and fliE genes. We inserted gRNA sequence into vector plasmid that containing Cas9 gene to construct pU6-Cas9 plasmid. This plasmid was then transfected into VNP20009 for selection and subsequent sequencing verification to confirm the deletion. (Chen et al., 2021)

flhD and fliE Gene Deletion Plasmid Construction

gDNA Sequence
• To verify the functionality of the BAX gene(BBa_K5363000), we amplified the BAX gene(BBa_K5363000) from the plasmid pSL886 PMusAFP-MusBax-(MS2)24-HHR-pA (Shao et al., 2024). Then, to mimic the plasmid-delivery function of killer Salmonella, we inserted the amplified gene into CD513B-SV40 to construct CD513B-Bax. The plasmid contains a CMV promoter that constitutively expresses genes in mammalian cells.

CD513B-Bax Plasmid Construction

• After constructing the CD513B-Bax plasmids, we transfected it into the B16-F10 cell line. Subsequently, we observed that the cells transfected with the CD513B-Bax plasmids exhibited a lack of continuous growth, and also a higher rate of cell death compared to the control groups, as determined by flow cytometer analysis. Additionally, Western blot analysis confirmed elevated BAX gene(BBa_K5363000) overexpression in the transfected cells, indicating that the overexpression of BAX protein led to the death of B16-F10 cells.

• We had uncertainties regarding the effectiveness of this method and were attentive to the specificity of the two strains of Salmonella. Therefore, there is a need to enhance the efficiency and safety of these two types of Salmonella to ensure improved functionality.

• Extensive literature reviews revealed the absence of an appropriate system for signal transduction in Gram-negative bacteria. Consequently, we required a promoter capable of facilitating bacterial expression upon invasion into cells. Researchers at the University of Massachusetts have utilized the PsseJ promoter(BBa_K5363013), which is activated intracellularly following the invasion of cancer cells by Salmonella, to deliver drugs to host cells (cancer cells) through intracellular self-lysis. (Raman et al., 2021)
• Moreover, to enhance the safety level, we added Synchronized lysis circuit (SLC) circuit to Salmonella VNP20009 to control its population under a safe level.

• To induce autonomous self-lysis of cancer cells following invasion by Salmonella, we constructed the plasmid pYX00004, which contains the PsseJ promoter and the φX174E (also called lyseE) gene(BBa_K5363015). φX174E is a gene that codes for the E protein of the Phix174 microvirus (Sinsheimervirus), which induces host cell lysis. Initially, the PsseJ promoter(BBa_K5363013) was synthesized based on its known nucleotide sequence. The lyseE(BBa_K5363015) gene was then amplified via polymerase chain reaction (PCR) from the phiX174 plasmid. Both the synthesized PsseJ promoter(BBa_K5363013) and the amplified lyseE gene(BBa_K5363015) were inserted into the pYX00004 plasmid vector. Consequently, the self-lysis plasmid pYX00004 was successfully constructed.

PsseJ-lyseE Plasmid (pYX00004) Construction
• To ensure biosafety, it is necessary to control the population density of both extracellular and intracellular helper Salmonella. Therefore, we constructed a genetic circuit to insert specific genes into the system, thereby achieving regulation of Salmonella population density.

LuxI-LuxR-lyseE Circuit Construction

• After constructing pYX00004 (PsseJ-mCherry)(BBa_K5363026) and pYX00004 (PsseJ-lyseE)(BBa_K5363027), we transform them and the original plasmid pYX00004 (pBAD-mCherry) into Salmonella, respectively. We verified that PsseJ promoter(BBa_K5363013) would not be activated when Salmonella is out of cells, and mCherry(BBa_K5363020) would be expressed when Salmonella is in human cells.

• In the third cycle, the successful construction of the self-lysis plasmid pYX00004 represented a significant advancement toward autonomous Salmonella-mediated killing of cancer cells. This cycle emphasized the necessity for a more sophisticated and tightly regulated gene expression system to attain efficient and specific targeting of cancer cells. The challenges encountered with the PsseJ-lyseE system(BBa_K5363027) offered valuable insights that steered the development of the Bio-switch approach in the fourth cycle.

• In eukaryotic cells, protein translation is initiated when a preinitiation complex, consisting of a 40S ribosome and initiation factors (eIFs), is recruited to the untranslated region (UTR) at the guanine-rich 5′ cap of mature mRNA molecules that have been exported to the cytoplasm. Cooperative activity by cap-binding protein eIF4E, RNA helicase eIF4A, central scaffolding protein eIF4G, and helicase enhancers eIF4B and eIF4H subsequently triggers RNA unwinding, ribosome attachment, and codon scanning.
• It is well known that the 3′ poly-adenine (poly(A)) tail enhances mRNA stability in living cells, but the underlying mechanism is still a subject of debate. Nevertheless, numerous studies support a "closed-loop" model in which poly(A)-binding protein (PABP) functions as another canonical eIF capable of simultaneously binding both poly(A) and eIF4G to induce a circularized mRNA configuration that is favorable for mRNA scanning, ribosome recycling, and protein translation.
• To engineer trigger-inducible translational devices, the incorporation of RNA-binding protein (RBP)-specific aptamers into the UTRs of target gene mRNA is a popular starting point, as it enables site-specific recruitment of RBP-containing regulatory proteins designed to control mRNA stability or eIF4E recruitment. With all these ideas, we constructed an intracellular signal-activated killing system, derived from Shao et al., 2024.

• To achieve efficient translational control over the expression of the effector gene (BAX)(BBa_K5363000) in human cells, we utilize synthetic translational initiation factors (STIFs) developed by Prof. Xie Mingqi’s team. (Shao et al., 2024) Following the "closed-loop" model of translation, it is believed that manipulating the mRNA circularization process can regulate translation.
• Firstly, an expression vector is required for a synthetic mRNA transcript containing a control region with RBP-specific aptamers in the 3'-UTR, with MS2-box motifs selected for this purpose. To reduce background activity from BAX(BBa_K5363000) mRNA translation independent of STIFs, trans-acting shRNA-binding sites are replaced with cis-acting hammerhead ribozyme (HHR)(BBa_K5363002) motifs. These HHR motifs should induce spontaneous self-excision of the natural poly(A) signal either before or immediately after nuclear mRNA export.
• Secondly, an antibody linked to rotaviral non-structural protein 3 (NSP3)(BBa_K5363007), which is an eIF4F-binding protein (eIFBP), is employed to recognize the intracellular signal. Thirdly, another antibody recognizing the intracellular signal is linked to bacteriophage-derived MS2 coat protein (MCP)(BBa_K5363003), which can bind to tandem repeats of cognate MS2-box motifs(BBa_K5363001). These three plasmids combine to form a killing system activated by STIF. The intracellular signal plays a crucial role in this system. If the two antibodies recognize the specific signal, forming a loop with BAX mRNA connected to NSP3-antibody-signal-antibody-MCP, translation of BAX protein will occur.
• Regarding the specific intracellular signal, EGFP(BBa_K5363011) is fused to NS3a(H1)(BBa_K5363005) for system verification. NS3a(H1):ANR represents a constitutive protein association pair, while LaG16(BBa_K5363004) is a high-affinity GFP nanobody. Consequently, ANR repeats ((ANR)8)(BBa_K53363006) are linked to NSP3(BBa_K5363007), LaG16(BBa_K5363004) is linked to MCP(BBa_K5363003), and the killing system coupled with STIF is activated.

• In the vector, NS3a(H1)(BBa_K5363005) was linked to EGFP(BBa_K5363011) to mimic the intracellular signal for validating our killing system. To optimize the efficiency of this process, it was crucial to initially establish a cellular environment conducive to strong NS3a(H1) expression.

Intracellular Signal Plasmid (pSL816 EGFP-NS3a(H1))(BBa_K5363010)
• A plasmid containing the BAX gene(BBa_K5363000) under the control of the Bio-switch was constructed. The BAX gene(BBa_K5363000) is positioned after the synthetic control region containing the RBP-specific aptamers.

Bax Plasmid (pSL886 PMusAFP-MusBax-(MS2)24-HHR-pA)

pSL582 PhCMV-(ANR)8-NSP3(B)-pA)

pSL776 PhCMV-MCP-LaG16-pA

• Before introducing the three bio-switch plasmids (pSL886, pSL582, pSL776) into B16-F10 cells, we first expressed the target protein (NS3a protein) in this cell line by transfecting pSL816 along with the expression-enhancing vector SB100. After 24 hours, we observed the fluorescence signal of the cells. With a transfection efficiency exceeding 70%, we then co-transfected the pSL886, pSL582 and pSL776 plasmids.
• Subsequently, we observed a significant difference in cell growth conditions between experimental groups. Specifically, cells expressing both NS3a protein and bio-switch showed a much higher death rate compared to control groups, which included cells without NS3a and those without three bio-switch plasmids. Additionally, Western blot method was employed to verify the expression levels of BAX gene(BBa_K5363000). We confirmed that the BAX gene(BBa_K5363000) is overexpressed in the presence of NS3a protein but has very low expression levels in its absence.

• The Bio-switch demonstrated high specificity and efficiency in controlling the expression of therapeutic genes. In vitro assays confirmed that BAX(BBa_K5363000) translation was strictly dependent on the presence of STIFs. (See more details in Results) Further analysis and experimentation are needed to gain a deeper understanding of the killing system and its application in cancer therapy. This study provides a foundation for future research in this area, and continued investigation may lead to valuable insights into the potential applications of the systems.

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