Difference between revisions of "Part:BBa K4986004"

Revision as of 11:59, 9 October 2023

Curcumin Production Systems (DCS and CURS)

Curcumin is a bright yellow chemical produced by plants of the curcuma longa species. It is often used as a condiment in food or traditional herbs, and it has attracted lots of attention by researchers in the aspect of cancer treatment. However, considering that the taste of ginger is hardly acceptable by many and even if it is accepted, the intake of ginger as a seasoning is low. Thus, we decided to design a system using Diketide-CoA synthase (DCS) and Curcumin synthase (CURS) genes which helps to catalyze the production of curcumin

Usage and Biology

We used the promoter T7 and BBa_B0015 terminator, which is a double terminator made up of both BBa_B0010 and BBa_B0010. The ribosome-binding site (RBS) we used is BBa_B0034. The synthesized DCS and CURS gene were cloned into the pET28a E coli expression vector and were transformed into Rosetta bacteria for expression.

Figure1 The design of the DCS and CURS.

Characterization

Bacteria transformed with DCS and CURS were cultured in LB medium containing appropriate antibiotics. When OD600 reached 0.6-0.8, 0.5 mM IPTG was added to induce protein expression. The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). Crude enzyme solution was obtained by sonication under ice bath conditions. Protein concentration was determined using the Bradford assay kit. For analysis of curcumin production, a reaction was conducted in 200 μL 100 mM PBS (pH 7.4) containing 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L crude enzyme solution. After a 1-hour reaction at 37°C, the samples were extracted with ethyl acetate and the organic layer was collected, dried, and resuspended in methanol. The wavelength of 420 nm, which is the maximum absorption wavelength of curcumin, was set on a spectrophotometer, and methanol was used as a blank for baseline correction. The sample solution was placed in a cuvette and its absorbance was measured. Finally, the curcumin concentration of the sample was calculated based on its absorbance using a standard curve prepared with known concentrations of curcumin standard as shown in Figure 3b and 3c.The Figure3A is principle schematic diagram of DCS and CURS.The results showed a significant increase in the production of curcumin by the engineered strains.

Figure2 Gel image of DCS and CURS.

Figure3 The result of expression of DCS and CURS.

In vitro anti-cancer activity of curcumin Due to the poor solubility of curcumin in water, a stock solution of 10 mM was prepared using DMSO (dimethyl sulfoxide) as an organic solvent. Specifically, under sterile conditions, an appropriate amount of curcumin powder was added to either DMSO or ethanol and thoroughly mixed until completely dissolved. The solution was then aseptically filtered through a 0.22 μm membrane and aliquoted into sterile 1.5 ml centrifuge tubes, stored at -20°C to maintain stability. To test the effect of curcumin on the CT26 colon cancer cell line in mice, CT26 cells were seeded at a density of 5000 cells per well in a 24-well plate. The complete culture medium consisted of DMEM basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37°C in a 5% CO2 environment until they reached 60-70% confluency. Curcumin at concentrations of 0, 5, and 10 μM (Solarbio, C7090, China) was used. Cell viability was assessed using the CCK8 assay at 24 hours after treatment. An aliquot of 100 µL of CCK8 solution was added to each well and incubated at 37°C for 3 hours. The absorbance at 450 nm was measured using an Microplate reader. The results, as shown in Figure 4C, indicated a significant decrease in CT26 cell viability after treatment with a curcumin concentration of 10 μM, demonstrating significant in vitro anti-cancer activity of curcumin.

Figure4 The results about in vitro anti-cancer activity of curcumin

Potential application directions

With this system, it helps to introduce curcumin ,which is a natural antioxidant and anti-inflammatory substance with wide-ranging potential applications. It has been found to have significant effects in cancer treatment, cardiovascular health, antibacterial and antiviral activities. Additionally, curcumin is used for treating arthritis, digestive issues, and skin diseases. In recent years, scientists have also discovered its effectiveness in improving brain function and cognitive abilities. In conclusion, curcumin has promising applications and provides a natural and effective option for health and wellness.

Sequence and Features