Formulation of L.B. medium:
(L.B. medium powder: 40g/L)
Tryptone: 10g/L
Yeast extract: 5g/L
Sodium chloride (NaCl): 10g/L
1. Weigh the ingredients of L.B. medium formulation according to the proportions and mix them in a conical flask. Add 1L of deionized water and stir with a glass rod until complete dissolution. Adjust the pH to 7.2 with 1 mol/L NaOH solution to obtain the L.B. medium stock solution.
2. For solid culture medium, agar powder, usually 15-20g/L, should also be added.
3. Put the dissolved medium solution into an autoclave and autoclave at 121℃ for 120min.
4. After sterilization is complete, remove the medium and place it at room temperature.
Recipient strain (EcN1917)
LB medium
0.1 M calcium chloride
Preservation
30% glycerol
1. Activate the strain by overnight incubation.
2. Transfer 100 μL of the overnight activated culture to 100 mL of liquid LB medium and incubate for 2 hours until OD600=0.6. Then, dispense the culture into 50 mL centrifuge tubes (25 mL each) and pre-cool at 4°C. 3. Centrifuge at 4000g for 10 minutes at 4°C, discarding the supernatant.
3. Centrifuge at 4000g for 10 minutes at 4°C, discard supernatant, and add 20 mL of 0.1 M CaCl2 to each tube (5 mL per tube). Incubate in an ice bath for 30 minutes.
4. Centrifuge again, discard supernatant, and add 1 mL of 15% 0.1 M CaCl2 (4 mL per 100 mL of initial culture solution). Dispense into 1.5 mL centrifuge tubes of 100 μL each. To preserve competent cells, add a mixture of calcium chloride and 30% glycerol (1:1) to each centrifuge tube and store at -80°C.
Log phase bacterial solution (OD600=0.6)
Glycerol solution (10%)
Plasmid solution
L.B. liquid medium
1. Take 1.5 ml of log phase bacterial solution with OD600=0.6 and place on ice for 10 minutes. Centrifuge at 1500g for 5 minutes at 4°C and discard the supernatant. Bacteria were resuspended using 1.5 ml of 10% pre-cooled glycerol solution and centrifuged again. Bacteria were resuspended using 750ul of pre-cooled glycerol solution and centrifuged again. Resuspend the organisms using 40ul of pre-cooled glycerol solution.
2. Add 1ul of plasmid solution and blow gently to homogenize.
3. Add the mixed solution into the pre-cooled dry electrotransfer cup, and put the cup into the electrotransfer apparatus for electroshock transformation.
4. Quickly add the transformed bacterial solution into 1ml L.B. liquid medium.
5. The bacteria were collected by centrifugation after 1 hour of incubation in a constant temperature shaker at 37℃ and resuspended using L.B. liquid medium.
6. Incubate in a constant temperature shaker at 37°C for 12 hours to amplify the plasmid.
Transformed E.coli cells
Normal E.coli cells
L.B. agar medium
Kanamycin
1. Synthesize blank control (normal E.coli cells) and Transformed E.coli cells respectively.
2. The experimental cells, blank control were coated on L.B. agar plates containing 100ug/ml kanamycin and incubated inverted for 16 hours.
Transformed E.coli cells.
1. Prepare appropriate amount of L.B. Liquid Medium as needed and preheat in a 37°C water bath.
2. Pick single colonies from the screening plate under aseptic conditions and inoculate into sterile 96-well plates or sterile test tubes containing pre-warmed L.B. Liquid Medium.
3. Place the inoculated medium in a constant temperature shaker at 37°C and incubate at an appropriate speed (usually 200-250 rpm).
Bacterial cultures
Total protein extraction kit
Protease inhibitor
PMSF
Cold lysate
BCA Protein Concentration Assay Kit
Western-blot reagents and equipment
Hyaluronidase ELISA Kit
0.22um filter membrane
1. Prepare the lysate: add 10ul of each protease inhibitor and PMSF to 1ml of cold lysate. mix well and set aside on ice.
2. weigh the organisms wet weight. Add the newly configured lysate according to the ratio (1g: 10ml) and mix well. Ultrasonic fragmentation was carried out under ice bath conditions with 3 seconds of sonication time and 3 seconds of interval for a total time of 45 minutes.
3. Centrifuge at 10,000 rpm for 20 min at 4°C. Collect the supernatant into a new sterile test tube.
4. Determine the protein concentration in the supernatant using the BCA Protein Concentration Assay Kit.
5. Western-blot assay: collect the logarithmic phase bacterial fluid and centrifuge at 10000rpm for 5 minutes. The supernatant was taken and precipitated for colony counting. The negative control group, positive control group, and the transformant endogenous reference protein GAPDH, Hyaluronidase as well as FlaB protein expression were detected using Western-blot assay with anti-His tag and anti-Flag tag as antibodies.
6. Determination of secretory proteins: The supernatant was filtered through 0.22um filter membrane. Secreted protein expression was determined according to the instructions of the hyaluronidase ELISA kit.
We employed three methods to measure the activity of hyaluronidase secreted by the engineered bacteria. These include extracellular matrix degradation simulation experiments, a hyaluronidase activity detection kit (turbidity method), and enzyme-linked immunosorbent assay (ELISA).
Supernatant of engineered bacteria cultured for 12 hours
methacrylic acid-modified hyaluronic acid
photoinitiator LAP
PBS buffer
hyaluronic acid activity detection kit
GC-MS systemhyaluronic acid ELISA kit
First, synthesize methacrylic acid-modified hyaluronic acid hydrogel. Dissolve the photoinitiator LAP in PBS buffer at a concentration of 0.5% (w/v) in advance. After heating the solution to 50°C, add 1% (w/v) methacrylic acid-modified hyaluronic acid monomer and stir in the dark at 1000 rpm for one hour until the hydrogel monomer solution is fully mixed. Then, pour 4 ml of the hydrogel monomer solution into each well of a 6-well cell culture plate and irradiate with a 400 nm blue light initiator for 1 minute until the hydrogel is completely cured. Cut the cured hydrogel into small pieces of equal weight and immerse them in the bacterial culture supernatant. Every 8 hours, remove the pieces and freeze-dry them to measure the dry weight of the hydrogel.
The determination of the secretion concentration of hyaluronidase follows the method provided by the ELISA detection kit. First, mix the bacterial suspension in the logarithmic growth phase thoroughly, then centrifuge at 12,000 g for 10 minutes to collect the supernatant. This process is repeated three times to collect the supernatants. Next, dilute the supernatant with a buffer in a gradient manner, and after adding the ELISA detection reagent, construct a standard curve to correlate the absorbance with the concentration of hyaluronidase. Finally, measure the time-series concentrations of hyaluronidase in the supernatant.
For measuring the activity of hyaluronidase, we used the turbidity method. Add 100 µl of a 0.015% (w/v) hyaluronic acid standard solution to a 96-well cell culture plate and incubate it for 10 minutes (at 37°C) with standard solutions of hyaluronidase at concentrations of 5 µg/ml, 10 µg/ml, 20 µg/ml, 40 µg/ml, and 80 µg/ml. The experimental group consists of the bacterial culture supernatant. Then, measure the OD600 and subtract the baseline absorbance from the blank group; a higher value indicates greater enzyme activity.
RAW264.7 cell line
fetal bovine serum
PBS buffer
The mouse macrophage cell line (RAW264.7) was purchased from the ATCC cell bank and cultured in DMEM medium containing 10% fetal bovine serum. The culture conditions were a 37°C incubator with 5% carbon dioxide.
RAW264.7 cell line
fetal bovine serum
PBS buffer
Co-culture the engineered bacteria with the chassis bacteria for 8 hours until reaching the logarithmic growth phase (OD600 between 0.4 and 0.6), then dilute by a factor of 1×10⁶ and add to the DMEM complete medium at 5% (v/v), replacing the macrophage culture medium. After co-culturing for 24 hours, scrape the macrophages on ice using a spatula, disrupt them with ultrasonic treatment, and determine the protein concentration using a BCA protein assay kit. Use the Western blot method to detect the expression of the reference protein β-actin and iNOS protein.