We divided our project into targeting section and therapeutic section, and conducted the following experiments to demonstrate these two parts.

Since the chemotherapy drug 5-FU is harmful to other normal cells in the human body, while 5-FC is harmless, we have decided to convert 5-FC into 5-FU in the intestines. Therefore, through genetic engineering technology, we have enabled E. coli Rosetta to express CDase. In the intestines, cytosine deaminase (CD) (Theys, Jan, et al. ) converts a non-toxic prodrug, 5-fluorocytosine (5-FC), into a toxic chemotherapy drug, 5-fluorouracil (5-FU), reducing the harm of 5-FU to the human body.

We cloned the CD gene (codA) into the pET23b plasmid, using the T7 promoter and B0015 terminator as gene circuit control system. We then transferred the constructed plasmid into E. coli Rosetta (host cells). We conducted experiments to assess the impact of 5-FU on the activity of CT26 cells cultured in a 24-well plate. Following the complete seeding of cells, various concentrations of 5-FU (IF0170, Solarbio) or PBS (as a control) were introduced. Subsequently, the cells were incubated at 37°C for a period of 36 hours. The evaluation of cell viability was carried out using the CCK8 assay kit (Beyotime, C0037). The results demonstrated a gradual decrease in the survival capacity of CT26 cells as the concentrations of 5-FU increased (Figure 2A).
Similarly, we investigated the effects of 5-FC on the activity of CT26 cells cultured in a 24-well plate. Once the cells were fully seeded, different concentrations of 5-FC (F123460, Aladdin) or PBS (as a control) were administered, followed by incubation at 37°C for 72 hours. Cell viability was assessed using the CCK8 assay kit (Beyotime, C0037). Notably, our findings indicated that there was no significant alteration in the survival ability of CT26 cells even as the concentrations of 5-FC increased (Figure 2D).
Subsequently, the CD enzyme activity of pT3-CD strain was determined. The engineering strain pT7-CD was cultured in LB medium. 10 mL of bacterial culture was taken, centrifuged to discard the supernatant, resuspended in 20 mM TrisHCl (pH 7.0) to collect cell pellets, and then subjected to ice pre-sonication treatment to collect crude enzyme solution. The protein concentration was determined using the Bradford method. The crude enzyme solution (5 mg/mL) was incubated with 15 mM 5-FC (F123460, Aladdin) for 12 hours at 37°C. Afterwards, 5-FU was extracted using methanol, the extract was dried using a vacuum centrifuge, and the sample was suspended in 50 μL methanol. 10 μL of the sample was added to 190 μL of 0.1 M HCl. A standard curve for 5-FU (SF8400, Solarbio) was prepared using a volumetric flask, and the sample was detected at 266 nm using a spectrophotometer. Figure 2C shows the activity of the CDase of the engineering strain pT7-CD.
We further tested the effect of engineered probiotics on the activity of CT26 cells. The engineered bacteria Rosetta were cultured in LB medium, and after 12 hours, the bacterial cells were collected. The OD600 was adjusted to 1 with PBS, and 10 mM 5-FC was added. The cells were incubated at 37°C for 12 hours. The supernatant was collected as a test sample and added to CT26 cells cultured in a 24-well plate. After incubation for 72 hours, cell viability was determined using the CCK8 assay kit (beyotime, C0037). It was found that the engineering strain pT7-CD significantly increased the production of 5-FU and decreased the survival ability of CT26 cells compared to the wild-type strain, as shown in Figure 2D.

We have decided to design/construct the INP-HIpA protein. Ice nucleation protein (INP) is a commonly used protein carrier in cell surface display technology (Dou, Jian-lin, et al.). It has a large region located on the outside of the cell, which can be used for the display of other proteins. We will use this protein to display the HIpA protein, which is a histone-like protein derived from Streptococcus and can bind to heparan sulfate proteoglycans (HSPG) on the surface of tumors (Boleij, Annemarie, et al.).

We used INP as the basis for fusing the hlpA gene to get the INP-HlpA protein. We used promoter pT7 and terminator B0015 as a gene circuit control system to express INP-HlpA and transform the recombinant plasmid based on pET23b into E. coli Rosetta.
Under the same conditions, we set up assessments for E.coli Rosetta and engineered bacteria E.coli Rosetta/pT7-hlpA. We put the bacteria together with two types of colorectal cancer cells (RKO and CT26) to observe the number of bacteria adhere the cancer cells.Quantitatively, our results showed that E.coli Rosetta without pT7-hlpA have about 42*10^5 CFU counts well of RKO cells and about 39*10^5 CFU counts well of CT26. In comparison, E.coli Rosetta with pT7-hlpA have about 82*10^5 CFU counts well of RKO cells lysate and about 82*10^5 CFU counts well of CT26 cells lysate. By calculation, fusion protein pT7-hlpA enhance the ability of E.coli to adhere to RKO cells by 195% and CT26 by 210%.

The basal transcription activity of the gene can be regulated within a specific temperature range by using a temperature-controlled promoter. We chose the pTcl42 promoter because its ideal activation temperature threshold is 42°C, and it has low expression levels at 37°C (Wu, Ming-Ru, et al.). MazF is a widely studied toxin-antitoxin (TA) system in Escherichia coli, and its mechanism of action is well defined (Tripathi, Arti, et al.). MazF can recognize ACA sequences and hydrolyze the phosphodiester bond at the first A position at either the 5' or 3' end, causing ribosome release from cleaved mRNA and preventing protein synthesis. Subsequently, improperly encoded polypeptides are released and degraded by intracellular proteases, leading to cell death. Therefore, we use the temperature-controlled promoter TC1-42 and mazF to induce bacterial lysis. In order to achieve temperature-induced self-lysis in the engineered strain, the pTcl42 promoter was placed upstream of the bacterial lysis gene mazF. Subsequently, the Tcl42 promoter and the mazF gene were cloned together into the pSB1A3 plasmid.
The successfully constructed plasmid was transformed into E. coli DH5a bacteria using heat shock method, and recombinant bacteria were screened on LB agar plates containing 100 μg/mL ampicillin. To evaluate the expression of the mazF gene at different temperatures, the transformed bacteria were cultured for 12 hours at 37°C and 42°C, and their OD600 values were measured using a spectrophotometer. Wild-type DH5a and DH5a carrying only the pTcl42 of pSB1A3 plasmid were used as controls. All experiments were performed in triplicate to ensure the reliability of the results. The results are shown in Figure 6A.
To evaluate the time dependence of the bacterial lysis system driven by the pTcl42 promoter at 42°C, a time-course test was performed. The engineered bacteria were added to a 96-well plate and cultured in a shaking incubator at 42°C. Every 2 hours, the OD600 values were measured using a microplate reader to assess bacterial growth. The results are shown in Figure 6B. All experiments were performed in triplicate to ensure the reliability of the results.
The experimental results showed that the temperature-inducible promoter was induced at 42°C, causing almost complete lysis of the bacteria within 12 hours. In the future, we plan to modify common suppositories to have a heating function. When it is necessary to completely kill the engineered bacteria, the heating function of the suppository will be activated to initiate the bacterial suicide system.

1. Boleij, Annemarie, et al. "Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells." Infection and immunity 77.12 (2009): 5519-5527.2. Dou, Jian-lin, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417.3. Theys, Jan, et al. "Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum." Cancer Gene Therapy 8.4 (2001): 294-297.4. Wu, Ming-Ru, et al. "A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS)." Nature communications 10.1 (2019): 2880.5. Tripathi, Arti, et al. "MazF-induced growth inhibition and persister generation in Escherichia coli." Journal of Biological chemistry 289.7 (2014): 4191-4205.