Experimental plan: Prepare culture medium and experimental consumables, activate plasmid-carrying strains

Experimental procedure:

1. Use of a clean bench: Open the clean bench and place centrifuge tube stand, test tube rack, marker pen, and alcohol for disinfection. Perform UV irradiation for 30 minutes.

2. Sterilization: Prepare Eppendorf tubes, PCR tubes, centrifuge tubes, and wrap them with gauze. Pack 2 boxes each of large, medium, and small tips, wrap the outer shell with parchment paper, and tie the tip boxes with string/rubber bands. Use pre-prepared LB culture media to fill 20 test tubes and prepare 50 mL x 2 bottles of LB liquid culture medium. Sterilize the above items at 121℃ for 15 minutes (this process takes about 1.5 hours). There was an experimental accident during the process of filling the liquid culture medium: the medium spilled on the clean bench, and it has been properly cleaned afterwards.

3. Activation of strains: Including 1 tube of E. coli DH5α/empty vector 1, 1 tube of DH5α/carrying fadL, fadD genes. 1 tube of E. coli DH5α/empty vector 2, 1 tube of DH5α/carrying phoA-hEGF-his gene. Open the clean bench, ventilate it, place the sterilized LB test tubes in the bench, light the alcohol lamp, burn the test tube mouth and stopper, add 100 μL of preserved bacterial solution to each tube, add 2.5 μL of Amp antibiotic stock solution, and burn the test tube mouth and stopper again. Seal the test tube mouth, tie the test tubes, and incubate overnight on a shaker (150 rpm, 37℃).

Experimental plan: Extract plasmids, PCR amplify target fragments

Experimental procedure:

1. Plasmid extraction: Extract 4 types of plasmids according to the plasmid extraction kit instructions: DH5α/empty vector 1, DH5α/carrying fadL, fadD genes. DH5α/empty vector 2, DH5α/carrying phoA-hEGF-his gene. After centrifuging the bacterial solution for 1 minute, collect the precipitate. Add p1 resuspension, invert and mix with p2, invert and mix with p3, and centrifuge for 10 minutes. Add 500 μL of BL reagent to the absorption column, discard the waste liquid, add the above centrifuged supernatant, wash twice with pw wash solution, each time centrifuging for 1 minute. Add 50 μL of EB solution to the membrane of the absorption column, centrifuge for 2 minutes. Use Nanodrop to measure the recovered DNA concentration and store the DNA at -20℃. Most people successfully extracted the plasmids in this step.

2. Dilute primers: Centrifuge the lyophilized 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 pheP, PAL, and two plasmid DNA fragments using high-fidelity enzyme Pfu. 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 in the previous step): 1 μL

dNTP Mixture 25 μL

Sterile water 20 μL

(2) Perform the following PCR amplification program:

98℃, 5 min

Repeat the following three steps for 30 cycles:

98℃ 10s

55℃ 5s

72℃ 1min/kb

72℃ 8 min

The plasmids, primers, and PCR products required for PCR are shown in the table below:

Experimental plan: Agarose gel electrophoresis, gel extraction, double digestion, and recovery

Experimental procedure:

1. Agarose gel electrophoresis: Add the above PCR products to the prepared agarose gel, add 5 μL DL 5000 DNA marker, and perform electrophoresis (120V, 20 min). After the electrophoresis is completed, observe using a gel imaging system, cut the desired DNA gel, and place it in a 2 mL centrifuge tube for labeling.

2. Gel extraction: Perform gel extraction on the PCR product DNA gel according to the instructions of the gel extraction 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 place the adsorption column back into the collection tube. Cut the single desired DNA band from the agarose gel and transfer it to a clean centrifuge tube. Add an equal volume of solution PN to the gel block and incubate at 50°C until the gel block is completely dissolved. Transfer the resulting solution to another adsorption column CA2, leave at room temperature for 2 min, and centrifuge for 1 min. Add 600 μL of wash buffer PW (check if ethanol has been added before use) to the adsorption column CA2, centrifuge for 1 min, repeat the wash twice, and air dry. Drop an appropriate amount of elution buffer EB onto the middle position of the adsorption membrane and leave at room temperature for 2 min. Centrifuge for 2 min and collect the DNA solution. Measure the recovered DNA concentration using a Nanodrop.

3. Double digestion: Perform double digestion on the extracted plasmid pET23b and amplified target DNA fragments using EcoRI and XhoI. Use 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 h.

4. Agarose gel electrophoresis: Add the digested products to an 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. The product was successfully extracted, but the quantity is small. Proceed to a second round of gel extraction.

Experimental plan: Activation of E. coli DH5α and Rosetta, preparation of competent cells

Experimental procedure:

1. Activation of bacterial 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, and burn the mouth of the test tubes and the caps. Inoculate 100 μL of stock culture into each test tube, burn the mouth of the test tubes and the caps again, seal the test tubes tightly, tie them together, and incubate overnight on a shaker (220 rpm, 37℃).

2. Preparation of competent cells:

(1) Open the laminar flow hood, place 1 mL, 200 μL, 5 mL, 2 mL, and 30 mL centrifuge tubes, and 2 x 50 mL LB medium for UV irradiation. Pre-cool 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a 4℃ refrigerator.

(2) Prepare DH5α and Rosetta competent cells as follows: Transfer the pre-activated DH5α and Rosetta bacteria to a pre-cooled centrifuge at 4°C, centrifuge at 5000 g for 10 min, discard the supernatant, and add 800 μL of solution.

Experimental Plan: Prepare solid agar plates with 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℃ under high pressure for 15 minutes. Allow to cool to around 60℃, add 150 μL of 100 mg/mL Ampicillin (Amp) antibiotic, shake well, and pour into disposable petri dishes (enough for approximately 10-15 plates).

2. Ligation reaction: Connect the vector and the target fragment through double enzyme digestion. The ligation system is as follows (Note: this is an empirical system, the optimal volume ratio of the vector/target fragment can also be calculated according to the instruction manual):

T4 DNA ligase 1 μL

10 x ligation buffer 2 μL

Linear plasmid 1 μL

Insert DNA 6 μL

Perform ligation at 16 ℃ for 1 hour using a PCR instrument. Obtain pET23b-insert and store the ligation product at -20℃.

3. Transformation of the ligation product: Add 10 μL of the above ligation product to 100 μL competent cells, incubate on ice for 30 minutes, heat shock at 42℃ for 60 seconds, incubate on ice for 5 minutes, add 1 mL LB liquid medium, and incubate at 37℃, 150 rpm for 1 hour for recovery. In a laminar flow hood, take 100 μL of the recovery solution and spread it onto an Ampicillin (Amp) resistant agar plate.

Experimental Plan: Colony PCR verification, expansion culture of positive clones, extraction of validated plasmids and transformation

Experimental Procedure:

1. PCR verification: Label 6 colonies on each plate. Take half of the colonies as templates and perform colony PCR using Taq enzyme. Primers are TF and TR. Follow the instructions for reaction system and program. Prepare a 50 mL agarose gel and use DL2000 DNA marker for electrophoresis. Presence of correctly sized bands indicates successful plasmid construction.

2. Expansion culture: Pick validated colonies and add them to 5 mL LB liquid medium, supplemented with 2.5 μL Ampicillin (Amp) antibiotic, for expansion culture.

3. Plasmid extraction: Follow the instructions of the plasmid extraction kit to extract two types of plasmids. After centrifuging the bacterial culture for 1 minute, collect the precipitate. Add p1 resuspension solution, invert to mix, add p2, invert to mix, and add p3, invert to mix, then centrifuge for 10 minutes. Add 500 μL of BL reagent to the column, discard the flow-through, add the above centrifuged supernatant, wash twice with PW washing solution, centrifuging for 1 minute each time. Add 50 μL of EB solution to the membrane of the column, centrifuge for 2 minutes. Measure the concentration of the recovered DNA using Nanodrop and store the DNA at -20℃.

4. Transformation: Transform the validated plasmid into expression-type Escherichia coli.

In this study, the fadL gene will be cloned into the pET23b vector to construct the recombinant plasmid pET23b-fadL. The plasmid will then be transformed into Escherichia coli Rosetta. The transformed E. coli Rosetta will be inoculated into LB medium containing antibiotics and cultured overnight. The bacterial culture (2 mL) will be collected by centrifugation at 10,000 rpm for 1 minute after adjusting the optical density at 600 nm (OD600) to 1.

A control sample with empty plasmid and Rosetta will also be prepared. The bacterial pellets will be resuspended in 2 mL of PBS buffer containing 100 μM palmitic acid (a long-chain fatty acid) and incubated for 1 hour to allow the bacteria to absorb the palmitic acid. The bacteria will then be centrifuged at 10,000 rpm for 1 minute, and the supernatant (extracellular sample) will be collected.

The bacterial pellets will be resuspended in PBS and sonicated at 75W for 5 minutes to dissolve the bacterial particles and release the intracellular contents (intracellular sample). Lipids will be extracted from both extracellular and intracellular samples using the following method: equal volumes of chloroform/methanol (2:1, v/v) will be added to the samples, mixed thoroughly, and allowed to stand for 10 minutes to transfer the lipids to the chloroform phase. The samples will then be centrifuged at 15,000 rpm for 10 minutes to separate the chloroform and aqueous phases. The chloroform phase containing the lipids will be collected and transferred to a new centrifuge tube.

The chloroform will be evaporated at 50°C in a fume hood and further dried for 30 minutes in a vacuum centrifugal evaporator. The dried lipids will be dissolved in 200 μL of lipid assay buffer by vortexing for 5 minutes. Palmitic acid standards will be prepared, and the palmitic acid content in the samples will be determined using a non-esterified fatty acid (NEFA) assay kit according to the manufacturer's instructions. The palmitic acid content in the samples will be calculated based on a standard curve. The data will be presented as mean ± standard deviation.

One-way analysis of variance (ANOVA) and Tukey's post hoc test will be used to determine the statistical significance of differences, with a p-value less than 0.05 considered significant.

The FadD gene will be synthesized and cloned into the pET23b vector for ligation, followed by transformation into Escherichia coli Rosetta. The palmitic acid content in both intracellular and extracellular samples will be determined using the method described above.

Expression of FadD resulted in a reduction of intracellular palmitic acid content.

FadL and FadD will be co-expressed in the pET23b vector and transformed into Escherichia coli Rosetta. The palmitic acid content in both intracellular and extracellular samples will be determined using the method described above.

The results showed a significant decrease in total palmitic acid content with the co-expression of FadL and FadD.

The pET23b vector carrying the phoA-hEGF-his fusion gene will be transformed into Escherichia coli Rosetta cells. The engineered bacteria will be inoculated into 50 ml of LB medium supplemented with ampicillin and incubated overnight. The next day, the bacterial culture will be collected by centrifuging at 10,000 x g for 1 minute. The supernatant will be transferred to a new centrifuge tube as the extracellular fraction sample.

The cell pellets will be washed with PBS to remove residual culture medium and then sonicated (150W, 1s sonication, 3s interval, a total of 20 minutes). The lysed cell suspension will be transferred to a new centrifuge tube as the intracellular content sample.

The protein solution will be mixed with 5 times the volume of reducing protein sample buffer at a ratio of 4:1 and denatured in a boiling water bath for 15 minutes. The proteins on a 15% polyacrylamide gel will then be separated at 120V using SDS-PAGE. Subsequently, the proteins on the SDS-PAGE gel will be transferred to a PVDF membrane (0.2 micrometer, 30 minutes) under low-temperature conditions. The membrane will be blocked with 5% skim milk (TBST) at room temperature for 1 hour.

The membrane will then be incubated overnight at 4°C with a mouse-derived anti-His tag antibody (1:1000, AH367, Beyotime). After washing the membrane with TBST three times for 10 minutes each, it will be incubated at room temperature for 1 hour with a horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) antibody (1:1000 dilution, A0216, Beyotime). The membrane will be washed three times with TBST for 10 minutes each. ECL A and B solutions will be mixed in a 1:1 ratio as instructed. After washing the PVDF membrane, it will be slightly dried on absorbent paper. Then the membrane will be placed on a chemiluminescent imager and covered with prepared ECL substrate, ensuring full immersion of the membrane. After 1 minute, the excess liquid will be removed using absorbent paper, and the membrane will be placed in the chemiluminescent imager, starting the chemiluminescent reaction according to the preset program.

The Western blot results showed a band at around 8 kDa in both the extracellular and intracellular fractions of Escherichia coli Rosetta cells, confirming the expression and secretion of the phoA-hEGF fusion protein. The signal peptide of phoA successfully directed the hEGF fusion protein to the periplasmic space. Future studies may focus on optimizing protein yield and purification.

ELISA results: The ELISA analysis also indicated that the phoA signal peptide successfully directed the hEGF fusion protein.

The phoA-hEGF fusion gene was synthesized and cloned into the pET23b vector, which was then transformed into Escherichia coli Rosetta. The culture supernatant and bacterial content samples were collected by ultrasonication and centrifugation. Escherichia coli Rosetta containing only the empty vector was used as a control. Human embryonic kidney 293T cells were seeded at a density of 5,000 cells per well in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, in a 96-well plate. The cells were cultured at 37°C and 5% CO2 in a humidified environment until they reached 60-70% confluency. Then, 10μL of bacterial content and filtered supernatant of the engineered bacterial strain were added to each well in DMEM medium. After 24 hours of incubation, cell viability was detected using the CCK8 assay; specifically, 10μL of CCK8 solution was added to each well and incubated at 37°C for 3 hours. The absorbance at 450nm was measured using an enzyme-linked immunosorbent assay reader. The average absorbance (a) values and standard deviations of each group were calculated, and the data were analyzed and plotted using GraphPad Prism. One-way ANOVA was used for statistical analysis of the data, followed by Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant.

The bacterial cellulose (BC) membrane was cut into 1 cm x 1 cm fragments and washed repeatedly with sterile PBS buffer (pH 7.4). E. coli Rosetta/p23b-phoA-hEGF was then inoculated into LB medium and cultured overnight at 37°C. The bacterial culture was collected, washed with PBS buffer, and the bacterial concentration was adjusted to OD600=1. Using a pipette, 100 μL of the bacterial culture was spread onto the BC membrane. BC membranes without bacterial culture were used as negative controls. The BC membranes with the bacterial culture were placed in sterile petri dishes, and an additional 100 μL of bacterial culture was directly spread on the petri dish as a positive control. The petri dishes were covered with sterile lids and placed in a constant temperature incubator for 3 hours at 37°C. Subsequently, the BC membranes containing bacterial suspension were carefully removed using sterile forceps and immersed in 0.9 mL of Luria-Bertani (LB) medium. A 10-fold serial dilution of the bacterial suspension was performed, and the diluted bacterial suspension was plated onto LB agar plates. The plates were incubated overnight at 37°C, and the formed colonies were counted to determine the colony-forming units (CFU) per milliliter of the original bacterial suspension. The results showed that E. coli Rosetta/p23b-phoA-hEGF can survive on the BC membrane. The negative control confirmed the absence of contamination during the experiment. These results demonstrate that the BC membrane can support the survival of E. coli Rosetta/p23b-phoA-hEGF, making it a suitable substrate for bacterial applications.

To evaluate the permeability of engineered bacteria on bacterial cellulose (BC) membranes, we first cut the BC membrane into 5 cm x 5 cm (depending on the actual experiment) fragments and washed them repeatedly with sterile PBS buffer (pH 7.4). Then, E. coli Rosetta/p23b-phoA-hEGF was inoculated into LB medium and cultured overnight at 37°C. The bacterial suspension was collected and washed with PBS buffer, and then the bacterial concentration was adjusted to OD600=1. Using a pipette, 100 μL of the bacterial suspension was spread onto the BC membrane, ensuring an even distribution. The BC membrane with the spread bacteria was placed in a sterile Petri dish and incubated at 37°C for 24 hours. The BC membrane was then imprinted on LB solid medium on both sides and incubated at 37°C for 12 hours. Finally, the growth of engineered bacteria colonies on the solid medium was observed.

LB solid medium was prepared, and wild-type Escherichia coli was activated. 50 μL of bacterial suspension was evenly spread on LB solid medium. The bacterial cellulose membrane was cut into fragments half the size of the Petri dish and washed with sterile PBS repeatedly. It was then soaked in a 50 μM silver nitrate solution for 3 hours to fully adsorb silver ions onto the bacterial cellulose membrane. The silver ion-infused bacterial cellulose membrane was then overlaid onto the LB solid medium inoculated with wild-type E. coli and placed in a 37°C constant temperature incubator overnight. The next day, the Petri dishes were taken out, and the growth of bacterial colonies was observed on the part covered by the silver ion-treated bacterial cellulose membrane and the uncovered part. The number and size of the colonies were compared to evaluate the antibacterial effects of silver ions on Escherichia coli.