Preparation of sensory cells

(1) Inoculate the laboratory preserved Escherichia coli BL 21 glycerol bacteria in LB plate, pick the newly activated single colony after overnight culture at 37°C and inoculate it in 20 ml of LB liquid medium, oscillate and culture it at 37°C for about 12 hours to the late stage of logarithmic growth, and then inoculate the bacterial suspension into 100 ml of LB liquid medium according to the ratio of 1:100, and oscillate and culture it at 37°C for 2~3h until the OD600≈0.5. OD600≈0.5.

(2)Transfer the culture solution into a centrifuge tube and cool it on ice for 10min, then centrifuge it at 3000 g for 10 min at 4°C.

(3) Discard the supernatant, gently suspend the cells with 10 ml of pre-cooled 0.05 mol/L CaCl2 solution, cool on ice for 15~30 min, and then centrifuge at 3000g for 10 min at 4°C.

(4) Discard the supernatant, add 4 ml of pre-cooled 0.05 mol/L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for a few minutes to form a sensory cell suspension.

(5) Dispense the receptor cells into small portions of 100 μl and store at -80°C.

Construction of recombinant vector

According to the expression system of E. coli, codon optimisation was performed to obtain the target sequence encoding the mature protein, and then the sequence was submitted to Suzhou Jinwei Zhi Biotechnology Co. for gene synthesis, and the synthesized target gene was constructed into the pET-28a(+) vector.

Construction of recombinant engineering bacteria

Plasmid extraction in E. coli was carried out according to the instructions of Tengen Plasmid Extraction Kit.

The transformation steps of E. coli plasmid were as follows: firstly, 100 μL of E. coli DH5α and E. coli BL21(DE3) were slowly melted on ice. Then 10 μL of recombinant plasmid was removed and mixed, flicked the wall of the tube to mix, and after standing on ice for 30 min, it was heat-struck at 42°C for 45-90 s. It was quickly placed on ice and incubated for 5 min, and 900 μL of LB medium was added for the recovery of the bacterium, which was oscillated for 2 h at 37°C, 220 rpm, then the recovered bacterial cells were centrifuged for 5 min at 4000 rpm, and the supernatant was removed from 900 μL, and the supernatant was removed from 900 μL. The remaining medium was blown and mixed and then spread on LB/Kanamycin solid plate, and incubated in 37℃ constant temperature incubator inverted overnight.

Colony PCR

(1) Design specific primers for different genes and send them to the company for synthesis.

(2) Pick the positive clones from the resistant plate, suspend them in 25 μL of double-distilled water, and boil them at 100°C for 10 min, then use 4 μL as the template, 8 μL of Taq PCR Master Mix (2×, with Blue Dye), 6 μL of ddH2O, 1 μL of forward primer, and 1 μL of reverse primer to carry out PCR amplification, and according to the different primer settings Different PCR systems were set up according to different primers. After PCR, 5 μL of the amplified product was added to the wells of 1% agarose gel electrophoresis, and the size of the bands amplified by the specific primers was observed to be consistent with the size of the genes in the gel imaging system.

Verification of positive clones

After single colonies were grown, they were picked into 3-5 mL of LB liquid medium containing Kanamycin and cultured overnight for 12-16 h. 1-2 mL of the bacterial solution was centrifuged and the supernatant discarded, and 100 μL of sterile water was added and mixed using a vortex shaker.

Then put into boiling water water bath for 10 min, cooled to room temperature, 5000 rpm/min, centrifuged for 1 min, and take the supernatant as the template of PCR system. Use the reaction system shown in Table 3.5 to carry out colony PCR amplification under the reaction conditions shown in Table 3.6 (the annealing temperature of step 3 in the reaction conditions is determined according to the annealing temperature of the primers for the target gene, and the extension time of step 4 is determined by the size of the target gene) to verify whether the recombinant is a positive clone, and detect the PCR product by 1% agarose gel electrophoresis after the end of the PCR if a bright single band appears at the size of the target gene.

After PCR, the PCR product was detected by 1% agarose gel electrophoresis, if a bright single band appeared at the size of the target gene, the transformation was successful. Mix the amplification solution of the successfully transformed positive clone with glycerol in equal proportion and store it in the refrigerator at -80℃ for spare use.

PCR reaction system

Conditions for PCR amplification reaction

Induction of expression by engineering bacteria

(1) Pick the clone with correct band size and inoculate it into LB liquid medium containing kanamycin 20mL for overnight culture to prepare seed solution, then transfer the seed solution into 300mL LB liquid medium containing corresponding antibiotics according to 1% inoculum volume, and cultivate it at 37°C, 200rpm until OD600=0.6~0.8, at the optimal IPTG inducing concentration, inducing time, temperature conditions to induce the expression of recombinant enzyme.

(2)Centrifuge the induced bacterial solution at 4°C, 4000×g for 10min, discard the supernatant, wash the precipitate with PBS buffer for three times, then add 1mg/mL lysozyme and freeze-thaw it repeatedly: 37°C for 5min, -80°C for 5min, for a total of three times, place the bacterial suspension in an ice bath, and use the cellular ultrasonic crusher to crush the bacterial cells, with the parameter setting as: power 200W The parameters were set as follows: power 200W, ultrasonic 2s, intermittent 3s, once 30min, repeat 3 times until the bacterial suspension was clarified, and finally centrifuged the bacterial suspension at 12000×g, 4°C for 10min, and the supernatant was collected as the crude enzyme solution, and then the crude enzyme solution and the precipitate were subjected to SDS-PAGE gel electrophoresis, to detect whether recombinant enzyme existed in the supernatant.

SDS-Polyacrylamide gel electrophoresis

(1) Preparation of Caulmers Brilliant Blue R-250 staining solution: weigh 1 g of 0.1% (W/V) Caulmers Brilliant Blue R-250 in a 1L beaker. Measure 250 ml of 25% (V/V) isopropanol into the above beaker and stir to dissolve. Add 100 ml of 10% (V/V) glacial acetic acid and stir well. Add 650 ml of deionised water and stir well. Remove the particulate matter with filter paper and store at room temperature.

(2) Preparation of Caumas Brilliant Blue decolourising solution: take 100 ml of 10% (V/V) glacial acetic acid, 50 ml of 5% (V/V) ethanol, 850 ml of distilled water and mix thoroughly.

(3) Check whether the electrophoresis gel plate is leaking: take two clean glass plates (one short plate, one long plate), align the two plates, put them on the main body of the gel maker, and then clamp them tightly with a wedge-shaped plate, which is consistent on both sides. Then put it on the glue maker and clamp it, then use a pipette to add water between the two sides of the glass plate and leave it for 10 min to see if it leaks night. If there is no leakage, pour out the water, if leakage, reassemble and continue to leakage check.

(4) Preparation of gel: firstly, mix 2.5 ml each of 10% lower gel solution A and solution B, and then add 30 μL of accelerant to instill them into the assembled good gel maker, and then use distilled water to seal the surface of the lower layer of the gel, after the lower layer of the gel solidified sufficiently (about 10-15 min), discard the distilled water, and absorb the residual distilled water with a filter paper, and then add the prepared upper layer of the gel solution (taking the 10% upper layer gel solution A, solution B each 1 ml mixed, and then add 12 μL of coagulant promoter), and then inserted into the comb, to be solidified (about 10-15 min) can be used.

(5) Pre-treatment of protein samples: Pipette gun to suck 40 ul protein samples to add 10 μL of the proportion of sampling buffer (5-fold dilution) Mixed, 100 ℃ water bath heating for 10 min, so that the protein denaturation, cooled to room temperature, centrifuged at 12,000 rpm for 5 minutes, the supernatant can be taken directly on the electrophoresis can be sampled.

(6) Electrophoresis: Use a pipette to add protein samples and Marker into gel wells sequentially and record the order. Use SWE Fast High Resolution Electrophoresis Buffer (G2081) for electrophoresis: 200 V, 30 min; stop electrophoresis when bromophenol blue reaches the bottom of the electrophoresis tank.

(7) Staining: remove the glue-making plate, cut the glue plate to cut the glue, and gently put it into the staining tank to be stained with Caulmers Brilliant Blue R-250 staining solution for 20-30 min.

(8) Decolorization: after the completion of staining, recover the staining solution, rinse the gel with pure water for 2-3 times, and then put it into the decolorization tank and add the Caumas Brilliant Blue decolorizing solution for decolorization until there is no background colour.

Pre-treatment of polyethylene materials

(1) Cut the required polyethylene film into square pieces of 3cm x 3cm size and weigh them with an analytical balance.

(2) Soak the film in 75% ethanol for 2h, then wash the film three times with autoclaved deionised water.

(3) It was placed in an ultraviolet irradiation in an ultra-clean bench for 1h, and then dried naturally overnight.

(4) In order to verify the adequacy of film sterilisation, the sterilised film was added to 20 ml of LB medium, and the medium was incubated on a shaker at 37°C, 200 r/min for 24 h. Observe whether the medium becomes turbid, if it becomes turbid, it means that the film is not sufficiently sterilised; if the medium is still clear and transparent, it means that the film is sufficiently sterilised. Sterilisation method of polyethylene microspheres is the same as that of polyethylene film.

Polyethylene degradation function verification

(1) Verification of PE film degradation by laccase and hydrolase After the induction of the expression of laccase and hydrolase engineering strains and pET-28a (+) of E. coli BL21 bacterial fluid for centrifugation (4 ℃, 5000 × g, 10min) and discard the supernatant, the use of 0.1 mol/L, pH 7 PBS buffer washed repeatedly for 3 times, add 1 mg/mL of lysozyme solution blowing suspension, and repeated freezing and thawing: 37 ℃ for 5min, -80 ℃ for 5min, -80 ℃ for 5min, -80 ℃ for 5min, -80 ℃ for 5min and -80 ℃ for 5min. -80 °C for 5 min, 3 times. The suspension was then placed in an ice bath, and the bacterial cells were broken using a cell ultrasonic breaker, with the parameters set to: power 300 W, ultrasonic for 2 s, and stop for 3 s, until the suspension became clear. Then centrifuged (4°C, 12000 × g, 30 min). Its supernatant was collected as crude enzyme solution, and the crude enzyme solution of E. coli BL21 with vector pET-28a(+) was used as a control. To ensure that the reaction system was not contaminated by bacteria during the mixing action of the crude enzyme solution with PE film, the above crude enzyme solution was filtered and decontaminated using 0.22 μm filter membrane in an ultra-clean bench before use and then incubated with the PE film sheet at 30°C and 150 rpm for 30 days, with three parallels in each group. Since the enzyme activity will decrease with the reaction time, it is necessary to add an appropriate amount of crude enzyme solution into the reaction system every day to ensure the normal progress of the reaction.

(2) Verification of the PE film degradation function of alkane monooxygenases According to the known methods in the literature, E. coli BL21 heterologously expressing alkane monooxygenase and E. coli BL21 with the empty vector pET-28a(+) as a control group (hereinafter referred to as E. coli BL21- pET-28a(+)) were inoculated with 5% inoculum and cultured in MSM medium with PE film as the only carbon source at 37 ℃ for 30 days (three replicates for each group). for 30 days (three replicates for each group)

Determination of weight loss and calculation of half-life of PE film

After 30 days of incubation, the PE films were washed in ultrasonic cleaner with 2% sodium dodecyl sulphate solution, 75% (v/v) alcohol and distilled water, each time for 30 min, and dried overnight at 60°C. The weight loss of PE films was determined using an analytical balance with an accuracy of 1 in 100,000 to determine the weight loss of the film. The weight loss was then calculated using the following formula.

W is the residual mass after treatment (mg) and WO is the initial mass (mg).

In order to further process the weight loss results and to visualise the reaction rates of the different enzymes involved in the polyethylene degradation reaction, the kinetic rate constants for the first stage of polyethylene degradation were calculated using the following formula.

W is the mean value of the mass of the treated film (g) and Wo is the mean value of the initial mass of the initial film. t is the reaction time (30 days). k is the primary kinetic rate constant and t is the reaction time (30 days). k is the primary kinetic rate constant and t is the reaction time (30 days).

The above rate constant k is taken into the following equation to calculate the degradation half-life of polyethylene (t1/2 ) in years.

Fourier Transform Infrared Spectroscopy (FTIR) Detection

The above cleaned and weighed polyethylene films were cut into 5 mm×5 mm pieces per group, and then the changes of functional groups on the surface of PE films were detected by FTIR spectroscopy (three parallels were sent to each group), the specific parameters were set as follows: the scanning range was 4000-500 cm-1 , the spectral resolution was 4 cm-1 , each sample was scanned for 32 times, and the data was processed by Origin 2024 software. Each sample was scanned 32 times and the data were processed by Origin 2024 software and the carbonyl index was calculated according to the FTIR results. The carbonyl index of FTIR spectra was used as an important index to quantitatively analyse the degree of oxidation of PE, and the carbonyl index will increase with the increase of oxidation degree. There are two common methods to analyse infrared spectra, namely, absorption peak area analysis and absorption peak height analysis. Since the absorption peak height analysis is easily affected by the samples and instruments, the carbonyl index (CI) was calculated using the absorption peak area, and the calculation formula is as follows:

In the above equation, Ac=o is the characteristic peak area of carbonyl group near 1 900~1 650 cm-1; Ac-H2 is the characteristic peak area of methylene group near 2960~2850 cm-1.

Depending on the enzyme verified the crude enzyme solution or engineered bacteria expressing alkane monooxygenase was mixed with polyethylene microspheres for 30 days using the same method. Then the same cleaning method was adopted as for the polyethylene films and then they were subjected to scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR).

Scanning electron microscopy (SEM) detection

After weighing, the polyethylene film was cut into small pieces of 2 mm x 2 mm and pasted on conductive double-sided carbon tape, while the polyethylene microspheres were directly pasted on conductive adhesive tape, and then the surface of the samples were sprayed with gold using an ion sputterer, and the changes in the morphology of the surface of the polyethylene film were observed using the secondary electron emission mode in a high vacuum at 25kV.

After obtaining the SEM results, the proportion of PE damage area in the images was calculated using ImageJ 1.54j software.