In the Contribution section, we present our experimental methods and results in the context of our project, which focuses on the treatment of colorectal cancer. We aim to outline how our work can contribute to future iGEM teams.

1. Growth of Bacterial Culture: We initiated our process when the optical density (OD) of the bacterial culture reached 0.6 at OD600, indicating optimal growth conditions with sufficient nutrients.
2. Low-Temperature Induction: We induced protein expression at 16°C.
3. pH Adjustment and Cell Resuspension: We adjusted the pH of the cell suspension to 7.4 using a 20 mM HCl buffer.
4. Ultrasonication Under Ice Bath: Ultrasonication was performed under ice bath conditions, with parameters set at 500 W power for 1 s followed by 3 s rest intervals, totaling 20 minutes of ultrasonication. This process aimed to break the bacteria and obtain bacterial lysates.
5. Cold Lysis: The cell lysate was kept on ice to prevent protein degradation by proteases.
6. Cold Centrifugation: Centrifugation at 4°C was performed to obtain soluble protein supernatant, minimizing bacterial debris.
7. 0.45 µm Filtration: Filtration through a 0.45 µm membrane was carried out to remove cell fragments and large particles.
8. Protein Purification: Purified protein was collected and stored for further use. A Ni column was equilibrated with 20 ml of Binding buffer.
9. Wash Buffer: We washed the column with 20 ml of wash buffer to remove impurities.
10. Protein Concentration: The protein concentrate was stored at -80°C for future use.

1. Reaction with H2O2 and Amplex Red Reagent: H2O2 and Amplex Red reagent (known to generate more fluorescent products) reacted in a 1:1 ratio, producing red fluorescence products, resorufin, with excitation and emission maxima at approximately 571 nm and 585 nm.
2. Measurement at 560 nm: The maximum absorption peak was detected at 560 nm using a spectrophotometer for optimal results.

Under conditions of 37°C and pH 7.4, the Km value of myrosinase  was determined to be 84.15 x 10^-6 M, with a Vmax of 15.51 µM/µM protein.
Optimal Conditions for myrosinase: The ideal conditions for myrosinase activity were found to be 37°C at pH 7.4.
In our experiments, we applied the methods mentioned above to identify the optimal conditions for myrosinase . We believe this information will contribute to future iGEM teams working on similar projects.
In the enzyme activity assay of myrosinase, we used the H2O2-Amplex Red system, which is frequently utilized in biological applications. By sharing our methods, we aim to contribute to future teams in the field of biology.
In the myrosinase enzyme activity assay, the crucial parameter is the enzyme reaction rate. The Michaelis-Menten equation and the fitted curve provide a valuable reference for other teams. This equation defines the reaction rate as the amount of glucose catalyzed per second per unit of protein, and it illustrates that a higher substrate concentration leads to a faster reaction rate.

The suicide gene we employed must be effective against E. coli while posing no harm to human cells. Thus, we utilized a lethal gene targeting the bacterial cell wall, making it suitable for future teams to use our developed suicide system.
We used the temperature-inducible promoter TCL42, which is activated at 42°C, making it ideal for our experiments at 37°C, where expression levels were minimal.
TCL-42 + SRRz Lysis Gene Testing
To validate the functionality of the TCL42 promoter at different temperatures, we designed an experiment in which the TCL42 promoter was placed upstream of the bacterial lysis gene SRRz. Initially, we cloned the TCL42 promoter and the SRRz gene into the pSB1A3 plasmid. We then transformed the recombinant plasmid into E. coli DH5α bacteria using heat shock transformation and selected them on LB agar plates containing 100 µg/mL ampicillin. To assess the expression of the SRRz gene at different temperatures, we cultured the transformed bacteria at 25°C, 37°C, and 42°C for 12 hours and measured their OD600 values. Wild-type DH5α and DH5α carrying only the pTcl42-pSB1A3 plasmid were used as controls. All experiments were performed in triplicate for reliability.
To evaluate the efficiency of the TCL42 promoter in driving the SRRz gene at 42°C, we cultured the bacteria on a shaker incubator at 42°C for 12 hours. We sampled 500 µL of the culture every 2 hours and measured the OD600 values to assess bacterial growth. Under the same conditions, we also cultured wild-type DH5α and DH5α carrying only the pTcl42-pSB1A3 plasmid as controls. All experiments were performed in triplicate to ensure the reliability of the results.

In conclusion, our team has successfully purified myrosinase  under optimized conditions, characterized its enzymatic activity, and developed a temperature-controlled suicide system for E. coli.  These contributions lay the groundwork for future iGEM teams and researchers in the field of biotechnology and synthetic biology.
Our purification process provides a detailed protocol for obtaining pure HRP, which can be invaluable to teams seeking to work with this enzyme.  Additionally, the enzyme activity assay and Michaelis-Menten analysis offer insights into the enzyme kinetics of HRP, aiding in the design of experiments and applications involving this enzyme.
The temperature-controlled suicide system, driven by the TCL42 promoter, offers a safe and effective method for controlling bacterial populations.  Future iGEM teams and researchers can utilize this system for a wide range of applications, ensuring the containment and controlled elimination of engineered bacteria.
We believe that our contributions will not only advance the field of synthetic biology but also facilitate the development of innovative solutions for various biological and medical challenges.  As we look ahead, we encourage iGEM teams to build upon our work, further expanding the possibilities of biotechnology.