Difference between revisions of "Part:BBa K5001001"

Latest revision as of 12:22, 12 October 2023

cytidine deaminase

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) 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.

Usage and Biology

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).

Figure 1 Design of the cd.

Characterization

Figure 2 Gel electrophoresis of the cd.

Figure 3 The experimental results related to CDase.

The effects of 5-FU on cell activity were examined in CT26 cells cultured in a 24-well plate. After the cells were fully seeded, different concentrations of 5-FU (IF0170, Solarbio) or PBS (control) were added, and the cells were incubated at 37°C for 36 hours. Cell viability was determined using the CCK8 assay kit (beyotime, C0037), and it was found that the survival ability of CT26 cells gradually decreased with increasing concentrations of 5-FU (Figure 3A).

We also examined the effect of 5-FC on the activity of CT26 cells. After the cells were fully seeded, different 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 determined using the CCK8 assay kit (beyotime, C0037), and it was found that there was no significant change in CT26 cell survival ability with increasing concentrations of 5-FC (Figure 3B).

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 3C shows the activity of the CDase of the engineering strain pT7-CD.

The recombinant 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 3D.

Potential application directions

This study demonstrates the capability of Cytosine Deaminase (CD) to convert the non-toxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic chemotherapy drug 5-fluorouracil (5-FU). The strategy of converting CD into a toxic chemotherapy agent at the tumor site has the potential to enhance the efficacy of 5-FU while reducing systemic side effects. In our future plans, we intend to utilize Escherichia coli Nissle 1917 (EcN) as the foundational microorganism. EcN, a probiotic strain of E. coli, is clinically employed primarily to treat inflammatory gastrointestinal disorders such as Crohn's disease and ulcerative colitis. EcN colonizes the human intestinal tract, preventing invasive pathogens from compromising the intestinal mucosa, thereby providing protection and repair to the mucosal barrier. EcN also plays a role in regulating the host's immune response, balancing the secretion of immune factors, enhancing host immune competence, and thus alleviating and treating inflammation. Recent research has revealed EcN's tumor-targeting potential, and when used in conjunction with chemotherapy drugs, it can enhance the anti-tumor effects of these drugs. Employing EcN to express the CD enzyme holds the promise of opening up new possibilities for treating colorectal cancer, one of the globally high-incidence and high-mortality cancers. Existing treatment methods, such as surgery, chemotherapy, and radiation therapy, often carry risks of side effects and recurrence. This engineered probiotic offers a novel therapeutic option that could potentially improve treatment outcomes, reduce side effects, and enhance the quality of life for patients. Its prospects are promising.

References

Theys, Jan, et al. "Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum." Cancer Gene Therapy 8.4 (2001): 294-297.

Sequence and Features