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Revision as of 14:37, 11 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 experiment demonstrates that cytosine deaminase (CD) can convert a non-toxic prodrug, 5-fluorocytosine (5-FC), into a toxic chemotherapeutic agent, 5-fluorouracil (5-FU). The strategy of converting CD into a toxic chemotherapeutic agent at the tumor site could enhance the efficacy of 5-FU and reduce systemic adverse effects. This may open up new possibilities for the treatment of colorectal cancer, one of the cancers with high incidence and mortality rates globally. Existing treatment methods such as surgery, chemotherapy, and radiation therapy often come with side effects and risks of recurrence. This engineered probiotic offers a new treatment option that has the potential to improve treatment outcomes, reduce side effects, and enhance the quality of life for patients. It has a promising future for development.

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