Week 1: Experimental Preparation
Experimental Plan: Prepare culture media and experimental consumables, activate plasmid-carrying bacterial strains.
Experimental Procedure:
1. Use of a clean bench: Open the clean bench, place centrifuge tube racks, test tube racks, marker pens, and disinfect them with alcohol. Perform UV irradiation for 30 minutes.
Translation: Week 1: Experimental Preparation
Experimental Plan: Prepare culture media and experimental consumables, activate plasmid-carrying bacterial strains.
Experimental Procedure:
1. Use of a clean bench: Open the clean bench and place centrifuge tube racks, test tube racks, marker pens. Disinfect them by alcohol and perform UV irradiation for 30 minutes.

2. Sterilization: Prepare Eppendorf tubes, PCR tubes, centrifuge tubes, and wrap them with gauze. Fill in 2 boxes each of large, medium, and small-sized tips. Wrap the outer shell with parchment paper and tie the tip box with string/rubber band. Prepare LB culture media in advance, distribute into 20 test tubes, and prepare 50 mL x 2 bottles of LB liquid media. Sterilize the above items at 121℃ for 15 minutes (this process takes about 1.5 hours). During the distribution of liquid media, there was an experimental accident: the media spilled on the clean bench, and it has been properly cleaned afterwards.
3. Bacterial activation: including 1 tube of E. coli DH5α/empty vector 1, 1 tube of DH5α/carrying pheP gene, 1 tube of E. coli DH5α/empty vector 2, 1 tube of DH5α/carrying PAL gene. Open the clean bench, ventilate it, place the sterilized LB tubes on the bench, ignite an alcohol lamp, burn the tube mouths and caps, add 100 μL of stock bacterial liquid to each tube, add 2.5 μL of Amp antibiotic solution, burn the tube mouths and caps again. Tighten the tube caps, bundle the tubes, and culture overnight on a shaker (150 rpm, 37℃).
Week 2: Template Extraction, Plasmid Construction Begins
Experimental Plan: Extract plasmids, PCR amplify target fragments.
Experimental Procedure:
1. Plasmid extraction: Extract 4 types of plasmids, DH5α/empty vector 1, DH5α/carrying l1 gene, DH5α/empty vector 2, DH5α/carrying tcl-42 gene, DH5α/carrying SRRZ gene, DH5α/carrying DCS,CUS gene, according to the instructions of the plasmid extraction kit. After centrifuging the bacterial liquid for 1 minute, collect the precipitate. Add p1 resuspension to each tube, invert and mix with p2, invert and mix with p3, then centrifuge for 10 minutes. Add 500 μL of BL reagent to the spin column, discard the flow-through, add the above centrifuged supernatant to the spin column, wash twice with PW wash solution, each time centrifuging for 1 minute. Add 50 μL of EB solution to the membrane of the spin column and centrifuge for 2 minutes. Use Nanodrop to measure the concentration of the recovered DNA and store the DNA at -20℃. 

2. Dilute primers
Centrifuge the powdered primers at 12000rpm for 1 minute. Add the appropriate amount of distilled water to dilute the primers to the desired concentration.

3. PCR amplification:
(1) Use high-fidelity enzyme Pfu to amplify the target gene and two vector DNA fragments. The reaction system is as follows:
Pfu 0.5 μL
10x Pfu buffer 5 μL
Primer 1 1 μL
Primer 2 1 μL
Template (plasmid extracted earlier): 1 μL
dNTP Mixture 25 μL
Sterile water 20 μL
(2) Perform PCR amplification program:
98℃, 5 min
Perform the following 3-step reactions for 30 cycles
98℃ 10s
55℃ 5s
72℃ 1min/kb
72℃ 8 min
The plasmid, primers, and products required for PCR are shown in the table below:
Week 3: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experimental procedure:
1. Agarose gel electrophoresis: Add the above PCR products to the pre-prepared agarose gel, add 5μL DL 5000 DNA marker, and perform electrophoresis (120V, 20 min). After electrophoresis, observe using a gel imaging system, cut the desired DNA gel, and place it in a 2 mL centrifuge tube for labeling.

2. Gel recovery: Perform gel recovery on the PCR product DNA gel according to the instructions of the gel recovery kit. Add 500 μL equilibration buffer BL to the adsorption column CA2, centrifuge for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back in the collection tube. Cut a single desired DNA band from the agarose gel and place it in a clean centrifuge tube. Add an equal volume of solution PN to the gel block, incubate at 50°C until the gel block is completely dissolved. Transfer the solution obtained from the previous step to another adsorption column CA2, leave at room temperature for 2 min, centrifuge for 1 min. Add 600 μL wash buffer PW to the adsorption column CA2 (check if ethanol has been added before use), centrifuge for 1 min, repeat the wash twice, and air dry. Add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane, leave at room temperature for 2 min. Centrifuge for 2 min and collect the DNA solution. Measure the concentration of the recovered DNA using Nanodrop.
3. Double enzyme digestion: Perform double enzyme digestion on the extracted plasmid pET23b and the amplified target DNA fragment using EcoRI and XhoI. Prepare a 5x20 μL reaction. The 20 μL digestion system is as follows:
DNA 1 μg
EcoRI 1 μL
XhoI 1 μL
10xY buffer 4 μL
H2O up to 20 μL
Digest at 37°C for 4 hours.
4. Agarose gel electrophoresis: Add the digested products to the agarose gel, add 5μL DL 5000 DNA marker, and perform electrophoresis (120V, 20 min). After electrophoresis, observe using a gel imaging system, cut the desired DNA gel, and place it in a 2 mL centrifuge tube for labeling. The product was successfully extracted, but the amount is small. Gel recovery will be performed a second time.
Week 4: Preparation of competent cells
Experimental plan: Activation of E. coli DH5α and Rosetta, preparation of competent cells
Experimental procedure:
1. Activation of strains: Including Escherichia coli DH5α and Rosetta. Open the laminar flow hood, ventilate, place sterilized LB test tubes on the bench, light an alcohol lamp, burn the test tube mouth and cap, add 100 μL preserved bacterial solution to each test tube, and burn the test tube mouth and cap again. Seal the test tube, bundle them together, and culture on a shaker until the OD reaches around 0.5.

Experimental procedure:
2. Preparation of competent cells:
(1) Open the laminar flow hood and place 1 mL pipette tips, 200 μL pipette tips, 5 mL pipette tips, 2 mL and 30 mL centrifuge tubes, and 2x50 mL LB culture medium for UV irradiation. Pre-chill 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a refrigerator at 4°C.
(2) Prepare DH5α and Rosetta competent cells according to the following steps: Transfer the pre-activated DH5α and Rosetta bacteria to the pre-chilled centrifuge at 4°C, centrifuge at 5000 g for 10 min, discard the supernatant, add 800 μL of 0.1 M CaCl2 solution, and incubate on ice for 40 min. Centrifuge again (4000 g, 10 min), discard the supernatant, add 100 μL of 15% glycerol and 0.1 M CaCl2 to each tube, resuspend the bacteria. Transfer the competent cells to 2 mL centrifuge tubes on ice, 100 μL per tube. Label and store the competent cells at -80°C.
Week 5  Ligation, Transformation
Experimental plan: Prepare solid agar plates containing antibiotics, perform ligation reaction, transform the ligation product
Experimental procedure:
1. Preparation of antibiotic plates: Prepare LB agar medium (add 4.5 g agar to 300 mL LB liquid medium), autoclave at 121°C under high temperature and pressure for 15 min. Allow to cool to around 60°C, add 150 μL of 100 mg/mL Amp antibiotic, shake well, pour into disposable petri dishes (enough for about 10-15 plates).
2. Ligation reaction: Perform ligation by double enzyme digestion of the vector and target fragment. The ligation system is as follows (note: this system is an empirical system, the optimal volume ratio of vector/target fragment can also be calculated according to the instructions):
T4 DNA ligase 1 μL
10x ligation buffer 2 μL
Linear plasmid 1 μL
Insert DNA 6 μL
Perform the ligation at 16°C in a PCR machine for 1 hour. Obtain pET23b-insert, and store the ligation product at -20°C.
3. Transformation of the ligation product: Add 10 μL of the above ligation product to 100 μL of competent cells, incubate on ice for 30 min, heat shock at 42°C water bath for 60 s, cool on ice for 5 min, add 1 mL of LB liquid medium, and incubate for 1 hour for recovery (37°C, 150 rpm). In the laminar flow hood, take 100 μL of the recovery solution and spread it on an Amp antibiotic plate with a sterile loop.

Week 6: Purification of myrosinase, Enzyme Activity Assay, and Optimal Reaction Conditions
First, clone myrosinase into the pET28a vector. Transform the ligation product into E. coli Rosetta (DE3) competent cells and verify by sequencing. Culture the engineered E. coli Rosetta strain in LB medium containing 50 μg/mL kanamycin. Once the bacterial growth reaches OD600=0.6, induce protein expression by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and incubate overnight at 16°C to reduce protein aggregation and inclusion body formation. Harvest the bacterial pellet by centrifugation at 10,000 g for 1 min. Then, resuspend the bacteria in 20 mM Tris-HCl buffer (pH 7.4). Use an ultrasonic homogenizer under ice bath conditions (500W, 1s sonication, 3s interval, 20 min) to obtain the cell lysate. Keep the cell lysate on ice and transfer it to a 50 mL centrifuge tube. Centrifuge at 12,000 rpm for 30 min at 4°C to obtain the soluble protein supernatant. Filter the supernatant through a 0.45 μm membrane to remove cell debris and large particles. Then proceed with the purification of myrosinase. Perform affinity chromatography purification using a nickel column (HisTrapTM HP, GE Healthcare, 5 mL). Store the purified protein in a new centrifuge tube for further use. Add 20 mL of binding buffer (10 mM imidazole) to the Ni column to equilibrate it. Load the cell lysate onto the column and repeat this step to load all the cell lysate onto the column. Wash the column with 20 mL of wash buffer (20 mM imidazole) to remove impurities, and then elute the target protein with 10 mL of elution buffer (300 mM imidazole). Collect the eluate in a new centrifuge tube. Concentrate the protein by adding 10 mL of the eluate into an ultrafiltration tube (Millipore, 10 kD) and centrifuge at 2,500 xg for 20 min at 4°C until the eluate is concentrated to around 1 mL. Add 9 mL of Tris-Base to remove imidazole and NaCl and centrifuge again to concentrate the solution to 1 mL. Repeat this step three times to obtain the concentrated target protein without imidazole and NaCl. Store it at -80°C for further use.
In this assay, glucose oxidase reacts with glucose to produce glucolactone and H2O2. In the presence of horseradish peroxidase (HRP), H2O2 reacts with Amplex® Red reagent in a 1:1 ratio to produce a red fluorescent product called resorufin. Resorufin has excitation and emission maxima at approximately 571 nm and 585 nm, respectively. This assay kit can be detected by fluorescence or absorbance measurements. 

Week 7 Testing the temperature-sensitive promoter Tcl-42
To validate the functionality of the temperature-sensitive promoter Tcl-42, we first fused it upstream of the mRFP reporter gene and cloned it into the pSB1A3 plasmid. Next, we transformed the recombinant plasmid into E. coli DH5α. The transformed strains were cultured for 12 hours at 37°C and 42°C. Using a plate reader, we measured the initial fluorescence intensity of mRFP at both temperatures and divided it by the OD600 to assess the activity of the Tcl-42 promoter.

Further testing the time dependency of the Tcl-42 promoter. Initially, all samples showed no fluorescence. As time progressed, due to the activity of the Tcl-42 promoter, samples at 42°C exhibited the fastest growth, while samples at 37°C and 25°C only showed minimal fluorescence leakage.

Week 8 Temperature-dependent testing of the Tcl-42+SRRZ thermosensitive promoter
To verify the functionality of the Tcl-42 promoter at different temperatures, we designed an experiment where the Tcl-42 promoter was placed upstream of the bacterial lysis gene SRRZ. First, we cloned the Tcl-42 promoter together with the SRRZ gene into the pSB1A3 plasmid. Next, the recombinant plasmid was transformed into E. coli DH5α bacteria using heat shock method and selected on LB agar plates containing 100 µg/mL Ampicillin. 
To evaluate the expression of the SRRZ gene at different temperatures, the transformed bacteria were cultured at 25°C, 37°C, and 42°C for 12 hours and their OD600 values were measured. Wild-type DH5α and DH5α carrying only the pTcl42 pSB1A3 plasmid were used as controls. All experiments were repeated three times to ensure the reliability of the results.

To evaluate the effectiveness of the Tcl42 promoter in driving the SRRZ gene at 42°C, the bacteria were cultured in a 42°C shaking incubator for 12 hours. Every 2 hours, 500 µL samples were taken and their OD600 values were measured to assess bacterial growth. Under the same conditions, DH5α carrying only the pTcl42 pSB1A3 plasmid and wild-type DH5α were also cultured as controls. All experiments were repeated three times to ensure the reliability of the results.

Week 9 - DCS and CURS catalyze curcumin production: reaction kinetics
To produce curcumin, DCS and CURS genes were synthesized and their codons were optimized for Rosetta. These two genes were then cloned into the pET28a expression vector and transformed into Rosetta bacteria for expression. The transformed bacteria were cultured in LB medium with appropriate antibiotics, and when the OD600 reached 0.6-0.8, protein expression was induced by adding 0.5 mM IPTG. The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4) and subjected to sonication under ice-bath conditions to obtain crude enzyme solution. Protein concentration was determined using the Bradford reagent kit. 
For the analysis of curcumin production, reactions were carried out in 200 μL of 100 mM PBS (pH 7.4) and contained 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L crude enzyme solution. After incubating at 37°C for 1 hour, the reaction mixture was extracted with ethyl acetate. The organic layer was collected, and the sample was dried and re-dissolved in methanol. The wavelength of the spectrophotometer was set to 420 nm, which is the maximum absorption wavelength of curcumin, and methanol was used as a blank for baseline correction. The sample solution was transferred to a cuvette, and its absorbance was measured. Finally, the curcumin concentration in the sample was calculated based on its absorbance using a standard curve prepared with known concentrations of curcumin standards.

In a reaction system containing 10-150 μM feruloyl-CoA and malonyl-CoA, and adding 40 mg/L crude enzyme in 200 μL of 100 mM PBS (pH 7.4), the reaction was carried out at 37°C for 5 minutes. The sample was extracted with ethyl acetate, and the organic layer was collected. The sample was then dried and re-dissolved in methanol. As mentioned before, the production of curcumin was measured. The reaction rate was calculated and fitted to the Michaelis-Menten equation. The Vmax was determined to be 286.2 nM/min, and the Km was 71.2 μM.

Week 10: In vitro anticancer activity, time dependence, and in vitro anticancer activity of curcumin
1. In vitro anticancer activity of sinigrinase
CT26 cells, a mouse colon cancer cell line, 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 an environment containing 5% CO2 until they reached 60-70% confluency. I1 sinigrinase produced by engineered bacteria at a concentration of 0.1 μM was incubated with 100 μM sinigrin (sinigrinase substrate). Samples were taken at 3, 24, and 72 hours after treatment, and cell viability was evaluated using the CCK8 assay. 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 enzyme immunoassay analyzer. The average absorbance and standard deviation of each group were calculated, and the data were analyzed and plotted using GraphPad Prism. One-way analysis of variance (ANOVA) was used to analyze the statistical differences in the data, followed by Tukey's post hoc test. A P-value less than 0.05 was considered statistically significant.
2. In vitro anticancer activity of curcumin
Due to the poor solubility of curcumin in water, a 10 mM stock solution was prepared using DMSO (dimethyl sulfoxide) as an organic solvent. Specifically, under sterile conditions, an appropriate amount of curcumin powder was added to DMSO or ethanol, thoroughly mixed until completely dissolved, sterile filtered using a 0.22 μm filter membrane, aliquoted into sterile 1.5 mL centrifuge tubes, and stored at -20°C to maintain stability. To test the effect of curcumin on CT26 cells, 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% FBS and 1% penicillin-streptomycin. The cells were cultured at 37°C in an environment containing 5% CO2 until they reached 60-70% confluency. Different concentrations of curcumin (0, 5, 10 μM) were used (Solarbio, C7090, China). Cell viability was assessed by the CCK8 assay 24 hours after treatment. 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 enzyme immunoassay analyzer.