Experiment Plan: Prepare culture media and experimental consumablesExperimental Procedure:1. Sterilization: It is recommended to wrap Eppendorf tubes, PCR tubes, and centrifuge tubes with gauze. Load large, medium, and small-sized tips separately, and wrap the shells with parchment paper. To fix the tip box, you can use ropes or rubber bands for bundling. At the same time, LB media that have been prepared should be aliquoted into 20 test tubes, and 2 bottles of 50mL LB liquid media need to be prepared. To ensure sterility, place the above items in a high-pressure autoclave for sterilization at 121°C for 15 minutes.

Experimental plan: Plasmid extraction, extraction of plasmid templates, PCR amplification of target fragments. Experimental procedure: 1. Activate strains: Including 1 tube of Escherichia coli DH5α/empty vector, 1 tube of DH5α/carrying CD gene, and 1 tube of Escherichia coli DH5α/inp-hlpA. Open the laminar flow hood, ventilate, place sterilized LB tubes in the hood, light an alcohol lamp, burn the tube mouth and tube cap. Add 100 μL of preserved bacterial solution to each tube, add 2.5 μL of Amp antibiotic stock solution, and burn the tube mouth and tube cap again. Tighten the tube cap, bundle the tubes, and incubate overnight on a shaker (150 rpm, 37℃). 2. Plasmid extraction: Extract the three types of plasmids (1 tube of Escherichia coli DH5α/empty vector, 1 tube of DH5α/carrying CD gene, and 1 tube of Escherichia coli DH5α/INP-hlpA) according to the instructions of the plasmid extraction kit.

2. Dilute the primersCentrifuge the powdered primers at 12000rpm for 1 minute, and dilute the primers to the appropriate concentration by adding the corresponding amount of distilled water.

3. PCR reaction:(1) Amplify the target gene empty vector, CD, and DNA fragments using high-fidelity enzyme Pfu. The reaction system is as follows:Pfu 0.5 μL10x Pfu buffer 5 μLPrimer 1 1 μLPrimer 2 1 μLTemplate (plasmid extracted in the previous step): 1 μLdNTP Mixture 25 μLSterile water 20 μL(2) Perform PCR amplification program:98℃, 5 minThe following 3 steps are repeated for 30 cycles:98℃ 10s55℃ 5s72℃ 1 min/kb72℃ 8 min

Experimental plan: Gel electrophoresis, gel recovery, double enzyme digestion, recoveryExperimental procedure:1. Gel electrophoresis: Add the above PCR product to the prepared agarose gel, add 5 μL 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 of equilibration buffer BL to the adsorption column CA2, centrifuge for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Cut the 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 piece, incubate at 50 degrees Celsius until the gel is completely dissolved. Transfer the obtained solution to another adsorption column CA2, let it stand at room temperature for 2 min, and centrifuge for 1 min. Add 600 μL of washing solution PW to the adsorption column CA2 (check if ethanol has been added before use), centrifuge for 1 min, repeat the washing process twice, and air dry. Add an appropriate amount of elution buffer EB to the center of the adsorption membrane, let it sit at room temperature for 2 min. Centrifuge for 2 min and collect the DNA solution. Measure the concentration of the recovered DNA using a Nanodrop.

3. Double Digestion: Perform double digestion on the extracted plasmid pET23b and amplified target DNA fragment using EcoRI and XhoI. Prepare a 20 μL reaction with a final volume of 20 μL. The components of the 20 μL digestion system are as follows:DNA - 1 μgEcoRI - 1 μLXhoI - 1 μL10x Y buffer - 4 μLH2O (add water) up to 20 μLIncubate the digestion reaction at 37°C for 4 hours.

4. Agarose Gel Electrophoresis: Add the digested products to an agarose gel and add 5 μL of DL 5000 DNA marker. Perform electrophoresis at 120V for 20 minutes. After the electrophoresis is complete, use a gel imaging system for observation. Cut out the desired DNA fragment from the gel and transfer it to a 2 mL centrifuge tube for labeling. The product extraction is successful but the quantity is low. Perform a second gel purification.

Experimental Plan: Activate E. coli DH5α and Rosetta, prepare competent cells.Experimental Procedure:1. Activation of cell strains: including Escherichia coli DH5α and Rosetta. Open the laminar flow hood, ensure proper ventilation, place sterilized LB tubes in the hood, ignite an alcohol burner, flame the tube openings and caps, add 100 μL of preserved bacterial stock solution to each tube, flame the tube openings and caps again. Close the tubes tightly, bundle them together, and incubate them overnight on a shaker (220 rpm, 37℃).Experimental Procedure:2. Preparation of Competent Cells:(1) Open the laminar flow hood and place 1 mL, 200 μL, 5 mL, 2 mL, and 30 mL centrifuge tubes, as well as 2 x 50 mL LB culture media inside. Perform UV irradiation. Pre-chill 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a refrigerator at 4℃.(2) Prepare DH5α and Rosetta competent cells using the following steps: Place the pre-activated DH5α and Rosetta bacteria in a pre-cooled centrifuge at 4℃, centrifuge at 5000 g for 10 minutes, discard the supernatant, add 800 μL of 0.1 M CaCl2 solution, and incubate on ice for 40 minutes. Centrifuge again (4000 g, 10 min), discard the supernatant, add 100 μL of 15% glycerol in 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℃.

Experimental Plan: Prepare solid agar plates with antibiotics, perform ligation reaction, transform the ligated product.Experimental Procedure:1. Prepare antibiotic plates: Prepare LB agar medium (add 4.5 g agarose to 300 mL LB liquid medium), autoclave at 121℃ under high pressure for 15 minutes. Allow it to cool to approximately 60℃, add 150 μL of 100 mg/mL Ampicillin antibiotic, mix well, and pour into disposable petri dishes (enough for 10-15 plates).

T4 DNA ligase 1 μL10x ligation buffer 2 μLLinear plasmid 1 μLInsert DNA 6 μLPerform the ligation reaction at 16℃ in a PCR machine for 1 hour. Obtain pET23b-insert, and store the ligated product at -20℃.3. Transformation of the ligated product: Add 10 μL of the ligation product mentioned above to 100 μL of competent cells, incubate on ice for 30 minutes, heat shock at 42℃ for 60 seconds, cool on ice for 5 minutes, and then add 1 mL of LB liquid medium. Incubate for 1 hour for recovery (37℃, 150 rpm). In the clean bench, take 100 μL of the recovery solution and spread it on an Ampicillin agar plate using a sterile inoculation loop.

Experimental Plan:1. Colony PCR verification: Label 6 colonies on each plate. Take half of the colonies as templates and perform colony PCR using Taq enzyme. Primers TF and TR are used. Follow the instructions for reaction system and program. Prepare a 50 mL agarose gel and perform electrophoresis using DL 2000 DNA marker. Presence of correctly sized bands indicates successful plasmid construction.2. Expansion cultivation: Pick the colonies that were verified correctly and add them to 5 mL of LB liquid medium. Add 2.5 μL of Ampicillin and perform expansion cultivation.3. Plasmid extraction: Extract the two types of plasmids according to the instructions of the plasmid extraction kit. After centrifugation of the bacterial culture for 1 minute, collect the pellet. Add p1 resuspension, invert and mix with p2, invert and mix with p3, and then centrifuge for 10 minutes. Add 500 μL of BL reagent to the column, discard the flow-through, add the supernatant obtained from previous centrifugation, wash the column twice with pw wash solution, each time centrifuging for 1 minute. Add 50 μL of EB solution to the membrane of the column, centrifuge for 2 minutes. Measure the DNA concentration of the recovered DNA using Nanodrop and store the DNA at -20℃. This step was successful for most people in extracting the plasmids.4. Transformation: Transform the verified plasmid 1 into expression type Escherichia coli.

Culture the seed in LB medium. Take 10 mL of bacterial culture, centrifuge to remove the supernatant, resuspend the cell pellet in 20 mM Tris HCl (pH 7.0), and collect the crude enzyme solution after ice pre-sonication treatment. Measure the protein concentration using the Bradford method. Incubate the crude enzyme solution at 37℃ with 5 mg/mL 5-FC (F123460, Aladdin) for 12 hours. Extract 5-FU with methanol, dry the extract using a vacuum centrifuge, and resuspend the sample with 50 μL of methanol. Take 10 μL aliquots of the samples and add 190 μL of 0.1 M HCl. Prepare a standard curve for 5-FU using a volumetric flask and measure the 5-FU content in the samples at 266 nm using a spectrophotometer.

1. Effects of 5-FC on cell viability: Culture CT26 cells in a 24-well plate. When the cells are confluent, add different concentrations of 5-FC (F123460, Aladdin) or PBS (control) and incubate at 37℃ for 72 hours. Measure cell viability using a CCK8 analysis kit (beyotime, C0037). The kit instructions are provided separately.2. Effects of pT7-CD on cell viability: Culture the recombinant bacteria Rosetta in LB medium. After 12 hours, collect the cells and adjust OD600 to 1 with PBS. Add 10 mM 5-FC and incubate at 37℃ for 12 hours. Centrifuge the cells.

1. Thaw the cells stored in liquid nitrogen at -196°C quickly to 37°C. Inoculate the cells into a 25 cm2 culture flask with 5 mL of complete DMEM medium (referred to as "complete medium") containing 10% fetal bovine serum (Gibco, Thermor Fisher) and 1% gentamicin. Resuscitate the CT26 mouse colorectal cancer cell line and the RKO human colorectal cancer cell line in a cell culture incubator at 37°C and 5% CO2. Once the cells form a monolayer, remove the DMEM medium. Digest the cells with 1 mL of trypsin for 1 minute, followed by 1 mL of complete medium to stop the digestion. Collect the cells and inoculate them into a 24-well plate. Use a microscope to observe the growth status of the cells, ensuring that they reach approximately 70%-80% confluency in the wells.Activate the recombinant engineered bacteria by inoculating them into LB medium. Adjust the OD600 to 0.6 on the following day. Inoculate 100 μL of the engineered bacteria into the wells of the 24-well plate (at this point, the wells are fully covered with cells). Incubate for 3 hours, then remove the supernatant and wash the wells three times with PBS.Add Triton X-100 (1% volume/volume ratio) to the wells and incubate at 28°C for 10 minutes to lyse the CT26 cells. Dilute the harvested cell lysate 10,000 times by gradient dilution, and spread it onto LB agar plates containing 50 mg/L ampicillin. Incubate for 12 hours, and then count the colony-forming units (CFUs) of the adherent bacteria on the next day. Repeat this assay three times to ensure reproducibility of the measurement.