Synthetic biology primarily involves the rational design and engineering of biological components, devices, or systems. Similar to other engineering fields, synthetic biologists follow a design-build-test iterative process to successfully develop innovative biological systems.
Through genetic engineering technology, our goal is to treat colorectal cancer. Here, we will provide a detailed description of the iterative process involved in our strain engineering. First, we conducted tests on CD enzymes, which can convert 5-FC into 5-FU, making it a key factor in cancer treatment. Next, we tested the engineered bacteria that overexpress INP-HlpA for their adhesive effects on colorectal cancer cells. Finally, we used a temperature-controlled promoter to regulate the expression of the mazF cleavage gene to ensure biosecurity. All of these genetic engineering processes were validated in the laboratory strain E. coli Rosetta. In the future, we hope to integrate these components into E. coli Nissle 1917. We chose Escherichia coli Nissle 1917 (EcN) as our engineered microbial host because it possesses non-pathogenic characteristics and an excellent genetic background. This provides a solid foundation for our applications. Furthermore, EcN has a stable genetic background, making it easy to manipulate genetically.

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 Nissle1917 to express CDase. In the intestines, cytosine deaminase (CD) converts a non-toxic prodrug (Theys, Jan, et al. ) , 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).

Test 1: The impact of 5-FU on cell activity
The impact of 5-FU on cell activity was assessed in CT26 cells cultivated in a 24-well plate. Following complete cell seeding, varying concentrations of 5-FU (IF0170, Solarbio) or PBS (control) were introduced, and the cells were incubated at 37°C for 36 hours. Utilizing the CCK8 assay kit (Beyotime, C0037), we determined cell viability. It was observed that the survival rate of CT26 cells gradually diminished with higher concentrations of 5-FU (Figure 3A).
Test 2: The impact of 5-FC on cell activity
Similarly, we investigated the effects of 5-FC on cell activity in mouse colorectal cancer cell line CT26 cultured in a 24-well plate. After full cell seeding, diverse concentrations of 5-FC (F123460, Aladdin) or PBS (control) were added, and the cells were incubated at 37°C for 72 hours. Cell viability was assessed using the CCK8 assay kit (Beyotime, C0037). Notably, there was no significant alteration in the survival capability of CT26 cells with increasing concentrations of 5-FC (Figure 3B).
Test 3: Measuring the CD enzyme activity of the pT3-CD strain
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 C shows the activity of the CDase of the engineering strain pT7-CD (Figure 3C).
Test 4:
We further tested the effect of engineered probiotics on the activity of CT26 cells. The engineered EcN 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 3D.

By conducting interviews with experts, we gathered valuable insights regarding the large intestine's length, typically spanning between 2 to 3 meters. It came to our attention that the initial generation of engineered strains faced challenges in accurately homing in on tumor regions. Following comprehensive team deliberations, we embarked on the development of a targeting module specifically designed to secure the engineered strains onto the surface of cancer cells. This strategic innovation is geared towards enhancing the precision and overall effectiveness of our treatment approach.

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%.

After improvement, the engineered strains are now capable of providing more effective treatment for colorectal cancer.However, considering the biosafety issue, we may need to introduce a suicide system to lyse the bacterial strains after functional expression.

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 plan to 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 8A.
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 8B. 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.

After three rounds of iterative design, the engineered strain has been able to effectively address our conceptual experiments.

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