1. Extract plasmid: extract 1 tube of pET23b and 1 tube of pSB1A3-insert according to the instructions of plasmid extraction kit.

(1) column balance: put the adsorption column CP3 into the collection tube, add 500 μ l equilibrium liquid BL to the adsorption column CP3, centrifuge 12000rpm for 1 minute, pour out the waste liquid and set aside.

(2) add 2mL bacteria to the 2mL centrifuge tube and centrifuge 12000rpm for 1 minute. The bacteria are deposited at the bottom of the tube, then pour out the liquid and use the liquid transfer gun to absorb the residual liquid.

(3) add 250 μ l of solution P1 and blow with a liquid transfer gun to mix well.

(4) add 250 μ l solution P2, turn it up and down gently for 8 times, and the liquid becomes clear and sticky.

(5) add 350 μ l solution P3 and turn it up and down gently for 8 times at once to produce white flocculent precipitation. centrifuge with 12000rpm for 10 minutes.

(6) transfer the supernatant (about 800 μ L) to the adsorption column CP3 in step (1), centrifuge with 12000rpm for 1 minute, pour out the waste liquid from the collection tube, and put the adsorption column CP3 back into the collection tube.

(7) add 600 μ L rinse solution PW,12000rpm to the adsorption column CP3 and centrifuge for 1 minute, then pour out the waste liquid.

(8) repeat steps (7).

(9) put the adsorption column CP3 into the collection tube and centrifuge for 2 minutes to remove the residual liquid from the adsorption column.

(10) uncover the adsorption column CP3 for 10 minutes and thoroughly dry the residual liquid in the adsorption column.

(11) put the adsorption column CP3 into a clean 1.5mL centrifuge tube, drop 80 μ L elution buffer EB in the middle, and centrifuge at room temperature for 2min and 12000rpm for 2 minutes.

2. PCRreaction

(1) using high fidelity enzyme Pfu DNA Polymerase to amplify the target gene from pSB1A3-insert.

Take out the 200 μ l PCR tube and add the following solution:

Centrifuge the liquid to the bottom of the pipe with a small centrifuge.

(2) PCR amplification program.

94 ℃, 3 min.

The following three steps are followed by 30 cycles.

94 ℃ for 30s.

55 ℃ for 30s.

72 ℃ 45s.

72 ℃ 10 min.

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

3. Preparation of 50 mL 1.2% agarose gel.

(1) dissolve 0.6 g agarose (Agarose) in 50 mL 1 X TAE buffer, use microwave oven to boil for 1-2 minutes, then boil twice for 10 seconds, cool down (not hot hands as the standard).

(2) prepare the dispensing board when cooling, and insert the 25 μ L comb into the 50 mL dispensing device.

(3) add 5 μ L 10 mg/mL EB solution, shake evenly, pour into the dispensing plate and wait for about 30 min to solidify.

4. Agarose gel electrophoresis.

(1) the above PCR products were added to the agarose gel and 5 μ L DL 5000 DNA marker was added for electrophoresis (150V DL 15 min).

(2) after the end of electrophoresis, the gel was observed by gel imaging system, and the DNA gel was cut and placed in the 1.5mL centrifuge tube.

5. Recovery of PCR products from glue.

The gel of DNA gel of PCR product was recovered according to the instructions of gel recovery kit (1 h), and the concentration of DNA was determined by Nanodrop.

(1) turn on the water bath or metal bath and adjust the temperature to 55 ℃.

(2) add 500 μ L solution to the glue block and bathe at PN,55 ℃ for 10-15 minutes, during which the centrifuge tube should be turned up and down until the glue block is completely dissolved.

(3) column balance: put the adsorption column CA2 into the collection tube, add 500 μ l equilibrium liquid BL to the adsorption column CA2, centrifuge 12000rpm for 1 minute, pour out the waste liquid and set aside.

(4) transfer the supernatant (about 800 μ L) to the adsorption column CA2 in step (1), centrifuge with 12000rpm for 1 minute, pour out the waste liquid from the collection tube, and put the adsorption column CA2 back into the collection tube.

(5) add 600 μ L rinse solution PW,12000rpm to the adsorption column CP3 and centrifuge for 1 minute, then pour out the waste liquid.

(6) repeat steps (5).

(7) put the adsorption column CA2 into the collection tube and centrifuge for 2 minutes to remove the residual liquid from the adsorption column.

(8) uncover the adsorption column CA2 for 10 minutes and thoroughly dry the residual liquid in the adsorption column.

(9) put the adsorption column CA2 into a clean 1.5mL centrifuge tube, drop 80 μ L elution buffer EB in the middle, and centrifuge at room temperature for 2min and 12000rpm for 2 minutes.

(10) the recovered products should be stored at-20 ℃.

6. Double enzyme digestion:

The extracted plasmid pET23b and the amplified target DNA fragment were digested by EcoRI and XhoI.

Take out the 200 μ l PCR tube and add the following solution:

Enzyme digestion at 37 ℃ for 1 hour

7. Connection reaction.

Take out the 200 μ l PCR tube and add the following solution connection system as follows:

Incubate the connection overnight with 16 °C to get pET23b-insert.

8. Conversion and connection products.

(1) turn on the water bath and adjust the temperature to 42 ℃.

(2) add 10 μ L ligated products to 50 μ L competent cells on ice, mix lightly and 20min in ice bath.

(3) Water bath at 42 ℃ for 90s, ice bath 2min, add 600 μ LLB liquid medium to the super clean table, culture 40min for resuscitation (37 ℃, 150rpm) [pay attention to aseptic operation].

(4) centrifuge with 8000rpm for 1 minute.

(5) in the super purification platform, the 500 μ L supernatant is sucked away with the gun head, and the remaining 100 μ L liquid is left, and the deposited bacteria are blown with the gun head to re-suspend it.

(6) add all the remaining liquid to the Amp antibiotic plate and spread the bacterial liquid evenly with an aseptic coating stick.

(7) the inverted plate was cultured overnight in the incubator at 37 ℃.

9. Culture medium and sterilization:

(1) 100mL LB liquid medium.

Tryptone peptone: 1g.

Yeast extract yeast powder: 0.5g.

NaCl:1g.

(2) 100 mL LB solid medium: (add 1.5g additional Agar).

(3) autoclave: 121C, 20min (the whole process is about 1.5h).

(4) inverted plate: pour 20mL around each petri dish (covered with a bottom), open the cover and blow 15min, and then use the sealing plate to seal the plate.

10. PCR verification:

A colony was selected as a template and Taq enzyme was used to PCR the colony. The primer was TF,TR, and the reaction system and procedure were carried out according to the instructions.

(1) using high fidelity enzyme Pfu DNA Polymerase to amplify the target gene from pSB1A3-insert.

(2) PCR amplification program.

94 ℃, 3 min.

The following three steps are followed by 30 cycles.

94 ℃ for 30s.

55 ℃ for 30s.

72 ℃ 45s.

72 ℃ 10 min

11. Shaking bacteria.

(1) add 5mLLB liquid medium (Amp+) to the shaker tube, pick up the recombinant colony with a small gun head, and shoot the gun head directly into the shake tube.

(2) add 5mLLB liquid medium (no resistance) to the shake bacteria tube, and put the E.coliRosetta strain 20 μ l into the shake bacteria tube.

12. Fluorescence microscope verification experiment:

(1) sample preparation: take appropriate amount (about 30 μ l) of Rosetta-pET23b- gene bacteria and wild type Rosetta onto the slide and cover it with the cover slide.

(2) turn on the power control box switch of the high pressure mercury lamp.

(3) insert the light shield to interrupt the light path.

(4) preheat 5-10min.

(5) put the slide containing the sample on the loading platform.

(6) choose the objective lens (in the order of low power first, then high power).

(7) rotate the turntable of the spectroscope assembly and select the spectroscope assembly that observes the mRFP (red fluorescence).

(8) adjust the focal length by coarse and fine spirals.

(9) turn on the computer connected to the microscope and click on the digital imaging system software to collect digital images.

13. The expression of fluorescent protein was detected by enzyme labeling instrument: 100 μ L recombinant bacterial liquid and E. coli Rosetta were added to 96-well plate and repeated for 4-6 times. The detection conditions of enzyme labeling instrument (mRFP excitation λ: 584nm/10 nm, emission λ: 607max 10 nm) were set and read. Use Excel to record data.

preparation before the experiment.

Synthesis of INP-PEAase sequence and INP-CBD sequence in Azenta Company.

Consult the map of pET23b vector and design the corresponding primers according to the target gene and vector information.

Configuration of LB liquid medium.

Configuration of PBS，1mM pNPB

Purchase and preparation of experimental consumables and experimental reagents

To study the expression and function of PETase in Escherichia coli Rosetta, we synthesized the PETase gene (Azenta life science, USA) and cloned it into the pET23b vector, allowing the target protein to be continuously expressed in E. coli Rosetta without the need for IPTG induction. The recombinant vector was transformed into E. coli Rosetta cells, and positive clones were selected on LB agar plates containing ampicillin. Positive clones were verified by DNA sequencing (Generalbiol, China). Positive clones were cultured overnight in LB medium, and cell pellets were collected by centrifugation and resuspended in Tris-HCl (pH 7.4). Cells were then lysed by sonication (150 W, sonication for 1s, interval 3s, for a total of 20 minutes) to obtain cell lysate. The total protein concentration in the lysate was measured using a Bradford reagent kit (Beyotime, China). Enzyme activity was measured using para-nitrophenyl butyrate (pNPB) as a substrate. A 100 mM pNPB stock solution (Merck, Germany) was prepared in acetonitrile. PETase activity was tested in a 100 μL reaction system: 1 mM pNPB, 50 mM Tris-HCl buffer (pH 7.4), 20 μL crude enzyme solution. The reaction was carried out at 30°C for 30 min. The release of para-nitrophenol ester from PETase cleavage of pNPB was measured at 405nm using a microplate reader. The content of para-nitrophenol ester was calculated based on a standard curve. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of para-nitrophenol ester per minute. The specific activity of PETase enzyme is defined as U/mg. All experiments were performed in triplicate, and data are presented as mean values with SD.

To improve the efficiency of engineered E. coli as whole-cell biocatalysts for PET degradation, the ice nucleation protein encoding gene INP was fused upstream of the PETase gene. The recombinant plasmid was transformed into E. coli Rosetta. Positive clones were cultured overnight in LB medium (37°C, 180 rpm), and 1 mL of bacterial culture was taken, adjusted to OD600=1, and centrifuged (10,000 rpm, 1 min) to collect cell pellets, which were resuspended in 1 mL PBS (pH 7.0). In 1 mL of bacterial suspension, 1 mM pNPB was added, and the reaction was incubated at 30°C for 30 min. The supernatant was collected by centrifugation, and the absorbance at 405nm was measured using a microplate reader. A standard curve was prepared using 0-1 mM para-nitrophenol ester dissolved in PBS. All experiments were performed in triplicate, and data are presented as mean values with SD.

E. coli Rosetta/pET23a-INP-PETase was cultured overnight in LB medium (37°C, 180 rpm), and 1 mL of bacterial culture was taken, adjusted to OD600=1. The culture medium was collected by centrifugation (10,000 x g, 1 min). The cell supernatant was transferred to a new centrifuge tube as an extracellular component sample. The cell pellet was washed with PBS to remove residual medium and lysed by sonication. The lysed cell suspension was transferred to a new centrifuge tube. Intracellular components and cell membranes were separated by centrifugation (e.g., 10,000 x g, 10 min). The supernatant was transferred to a new centrifuge tube as an intracellular component sample. The cell pellet was retained as a cell membrane sample. The cell pellet was resuspended in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100. The suspension was gently shaken or vortexed to dissolve the cell membrane proteins. Insoluble material was removed by centrifugation at 14,000 x g for 30 min. The supernatant was transferred to a new centrifuge tube as a cell membrane protein sample. The concentrations of extracellular, intracellular, and cell membrane proteins were measured using a Bradford reagent kit. Enzyme activity was measured using para-nitrophenyl butyrate (pNPB) as a substrate.

In order to enable E. coli to adhere to the cellulose membrane, the INP-CBD sequence was synthesized and cloned into the pET23b vector. The recombinant plasmid pET23b/INP-CBD was then transformed into E. coli Rosetta. Subsequently, the adhesion effect of the recombinant strain to bacterial cellulose was tested. The purchased sterile bacterial cellulose membrane (BC membrane) was cut into 5 cm x 5 cm pieces. The recombinant E. coli was cultured overnight in 5 mL LB medium containing 100 μg/mL ampicillin. The following day, bacterial pellets were collected by centrifugation, washed with PBS (pH 7.4), and the recombinant E. coli was resuspended to adjust the bacterial OD600 = 0.5. The recombinant E. coli suspension was then incubated with the BC membrane for 1 hour. Afterwards, the membrane was gently rinsed with 10 mL PBS to remove non-specifically adhered cells, and the rinse solution was collected. The rinsing solution was subsequently diluted in a gradient and spread onto LB AGAR plates containing ampicillin.. For comparison, wild-type E. coli was processed in the same way. After 24 hours of incubation, we calculated the CFU/mL for each sample. By comparing the non-specific adhesion of the INP-CBD fusion protein E. coli with that of the wild type on the bacterial cellulose membrane, we evaluated the adhesive effect of INP-CBD. Finally, the results were statistically analyzed using the student t-test.

Statistical Analysis

All experiments were repeated three times and the results are presented as means ± SD. The study data were statistically analyzed using Student's t-test (two-tailed, unpaired). Significance was denoted as *P < 0.05, **P < 0.01 and ***P < 0.001.