Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain

Experimental procedure:

1. Use of ultra-clean table: open the ultra-clean table, place the centrifuge tube plate, test tube rack, Marker pen, alcohol disinfection and sterilization, and conduct UV irradiation for 30 minutes.

2. Sterilization: Prepare Eppendorf tubes, PCR tubes, and centrifuge tubes, and wrap them with gauze. Fill 2 boxes each of large, medium, small, and gun heads, using Velcro paper to wrap the shell, and using string/rubber band to bind the gun head box. Using pre-prepared LB medium, divide 20 tubes into 50 mL x 2 bottles of LB liquid medium. The above items were sterilized using 121 ° C for 15 min (this process was about 1.5 h). An experimental accident occurred during the filling of liquid medium: the medium was spilled on the ultra-clean table, and it was cleaned up properly afterwards.

3. Activated strains: 1 tube of E. coli DH5α/ empty vector,1 tube of E. coli DH5α/ AL2 gene,1 tube of E. coli DH5α/bgls, 1 tube of DH5α/ arabinose promoter gene, and 1 tube of DH5α/ SRRz cleavage gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the orifice and plug of the test tube, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the orifice and plug of the test tube again. After sealing the tube, the tube was bundled and incubated on a shaker overnight (150 rpm, 37℃).

Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR

Experimental procedure:

1. Extract plasmid: According to the instruction of plasmid extraction kit, four plasmid tubes of Escherichia coli DH5α/ empty vector, one tube of DH5α/ carrying AL2 gene, one tube of Escherichia coli DH5α/bgls, one tube of DH5α/ carrying arabinose promoter gene, and one tube of DH5α/ carrying SRRz cleavage gene were extracted and centrifuged for 1min, then the precipitate was collected. p1 was added and resuspended, p2 was mixed upside down, p3 was mixed upside down, and then centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.

2. Dilute the primers

The dry powder primer was centrifuged at 12,000 RPM for 1min, and the corresponding amount of distilled water was added to dilute the primer to the appropriate concentration

3.PCR reaction:

(1) The empty vector of target gene, AL2 gene,bgls, arabinose promoter gene were amplified by high fidelity enzyme Pfu, and the DNA fragment of the gene was cleaved by SRRz. The reaction system is as follows:

Pfu 0.5 μL

5 μL of 10xPfu buffer

Primer 1 1 μL

Primer 2 1 μL

Template (plasmid extracted above) : 1μL

25μL dNTP Mixture

20 μL of sterilized water

(2) Perform the PCR amplification procedure

At 98 ° C for 5 min

The following 3-step reaction was performed for 30 cycles

98℃ 10s

55℃ 5s

72℃ 1min/kb

72℃ 8 min

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

Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery

Experimental procedure:

1 Agarose gel electrophoresis: The above PCR products were added to a previously prepared agarose gel with 5μL DL 5000 DNA marker and subjected to electrophoresis (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled.

2. Gel recovery: The PCR product DNA gel was recovered according to the instructions of the gel recovery kit

Add 500 microliters of equilibrium solution BL to the adsorption column CA2 and centrifuge for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single band of target DNA was cut from the agarose gel into a clean centrifuge tube. Add an equal volume of solution PN to the gel block and place in a water bath at 50 ° C until the gel block is completely dissolved. The solution obtained in the previous step was added to another adsorption column CA2, placed at room temperature for 2 min, and centrifuged for 1min. Add 600 microliters of bleach solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge for 1min, repeat rinsing twice, and let dry. Add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane and place it at room temperature for 2 min. The DNA solution was collected by centrifugation for 2min. The recovered DNA concentration was determined using Nanodrop.

3 Double digestion: EcoRI and XhoI were used to double digest the extracted plasmid pET23b and the amplified target DNA fragment. 5x20 μL reaction. The 20 μL digestion system is as follows:

1 μg of DNA

1 μL of EcoRI

1 μL XhoI

4 μL 10xY buffer

H2O up to 20μL

The enzyme was digested at 37 ° C for 4h

4 Agarose gel electrophoresis: The digested product was added to the agarose gel, 5μL of DL 5000 DNA marker was added, and electrophoresis was performed (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled. The product was extracted successfully, but the amount was small. After that, the second gum recovery was carried out.

Experimental plan: E. coli DH5α and Rosetta were activated to prepare competent cells

Experimental procedure:

1. Activated strains: including E. coli DH5α and Rosetta. Turn on the ultra-clean table, ventilate, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube mouth and test tube stopper, connect 100 μL of preserved bacteria solution into each test tube, and burn the test tube mouth and test tube stopper again. The test tube was closed, bundled, and incubated on a shaker overnight (220 rpm, 37℃).

Experimental procedure:

2. Preparation of competent cells:

(1) Open the ultra-clean stage, put in 1 mL tip, 200 μL tip, 5 mL tip, 2mL and 30 mL centrifuge tubes, and 2 x 50 mL LB medium for UV irradiation. The 0.1 M cacl 2 and 15% 0.1 M cacl 2 solution were pre-cooled in a refrigerator at 4 ° C.

(2) DH5α and Rosetta competent cells were prepared as follows: pre-activated and repacked DH5α and Rosetta bacteria were precooled in a centrifuge at 4 ° C, centrifuged at 5000 g for 10 min, the supernatant was discard, and 800 microliters of 0.1 M CaCl2 solution was added to an ice bath for 40 min. After centrifugation again (4000 g, 10 min), the supernatant was discarded, and 100 microliters of 15% glycerol 0.1 M CaCl2 were added to each tube to resuspend the bacteria. Aliquots of competent cells into 2 mL centrifuge tubes of 100μL per tube were dispensed on ice. After labeling, the competent cells were stored in the refrigerator at -80℃.

Experimental plan: Prepare solid AGAR plates containing antibiotics, perform ligation reactions, and transform ligation products

Experimental procedure:

1. Preparation of antibiotic plates: LB solid medium (4.5 g AGAR added to 300 mL LB liquid medium) was prepared and sterilized at 121 ° C for 15 min by autoclave. After cooling to about 60℃, add 150 μL of 100mg/mL Amp antibiotic, shake evenly, and pour into the disposable Petri dish (about 10-15 plates).

2. Ligation reaction: double enzyme digestion of the vector and the target fragment was connected. The ligation system is as follows (Note: This system is an empirical system, and the optimal linking vector/target fragment volume ratio can also be calculated according to the instructions) :

T4 DNA ligase 1 μL

2 μL of 10 x ligation buffer

1 μL of Linear plasmid

Insert 6 μL of DNA

Connect for 1h at 16 ° C using a PCR apparatus. pET23b-insert was obtained, and the ligation product was stored at -20 ° C.

3. Transformation of ligation products: 10 μL of the above ligation products were added to 100 μL competent cells, followed by an ice bath for 30 min, a water bath at 42 ° C for 60 s, and an ice bath for 5 min. Then, 1 mL LB liquid medium was added and incubated for 1 h for recovery (37 ° C, 150 rpm). In an ultra-clean stage, 100 μL of recovery solution was taken and the Amp antibiotic plate was coated with a sterile coating rod.

Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the correct plasmid and transformation

Experimental procedure:

1.PCR verification: Six colonies were labeled on each plate. Half of the colonies were picked and used as templates for colony PCR using Taq enzyme. The primers are 1,2. Reaction systems and procedures were carried out according to the instructions. Configure a 50 mL agarose gel, using DL 2000 DNA marker, and perform electrophoresis. The correct size of the band indicates that the plasmid was successfully constructed

2. Expanded culture: The correct colonies were selected and added to 5 mL LB liquid medium, and 2.5 μL Amp antibiotic was added to expand the culture.

3. Plasmid extraction: Two kinds of plasmids were extracted according to the instruction of plasmid extraction kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.

4. Transformation: Verify the correct plasmid 1 transformation into expression Escherichia coli.

Experimental procedure:

1. Verification of alginate lyase AL2:

The expanded bacterial solution was centrifuged to collect cell precipitates and resuspended in Tris-HCl (pH 7.4). Cell lysates were then obtained by lysis by ultrasonic fragmentation (150W, 1s sonication, 3s interval, 20 min in total). To verify the function of alginate lyase, 0.9 mL of substrate solution (0.5% (w/v) of alginate (Solarbio), 50mM Tris-HCI buffer, 200mM NaCl, pH 7.3) was mixed with 0.1 mL of cell lysate. The cells were incubated at 37°C for 30 min before sampling. The 3, 5-dinitrosalicylic acid (DNS) method was used to determine the concentration of reducing sugar. Absorbance at 540nm was recorded to calculate activity. One enzyme activity unit (U) was defined as the amount of enzyme required to release 1μmol of reducing sugar per minute. Protein concentration was determined using Bradford (using fetal bovine serum protein as a standard), and the specific activity of alginate lyase was defined as U/mg. All experiments were performed in triplicate and data will be expressed as mean plus SD.

Controls were experiments performed using untransformed E. coli Rosetta. The activity of the control group was 5 U/mg, which may be due to the weak effect of other cell components on the brown algae gum or the background noise of the experiment. Under our experimental conditions, the specific activity of the crude enzyme solution of alginate lyase was 323 U/mg. This means that each milligram of the crude enzyme solution is capable of releasing 323 micromoles of reducing sugar per minute. This result demonstrated the efficient activity of alginate lyase in our engineered bacteria and provided strong support for further application.

2. Effect of temperature on alginate lyase:

To determine the optimal reaction temperature for alginate lyase, cell lysates were diluted using Tris-HCl buffer pH 7.4 and mixed with 0.5% (w/v) alginate solution. After incubation at 25 ° C, 37 ° C, and 45 ° C for 30 min, the concentration of reducing sugar was determined using the 3, 5-dinitrosalicylic acid (DNS) method.

3. Effect of PH on alginate lyase:

To determine the optimal reaction pH for alginate lyase, we mixed cell lysates with 0.5% (w/v) alginate solution using citrate buffer (10 mM) at pH 6.1 and Tris-HCl buffer (10 mM) at pH7.4, 8.5, and 10.2. After incubation at 37 ° C for 30 min, the concentration of reducing sugar was determined using the 3, 5-dinitrosalicylic acid (DNS) method. Buffer pair groups were set and no enzyme was added, only substrate and buffer were added. This measures the background absorbance of each buffer and subtract this background value from the experimental group's data.

Note: When Tris is reacted with HCl, Tris-hcl can be obtained. The pH of the Tris-HCl buffer ranges from 7.0 to 11. The citrate buffer uses a reaction of sodium citrate and citric acid ranging from 3 to 6.2.

Experimental procedure:

1. Determination of cellulase Bgls activity:

The engineered strains were grown in LB medium at 37°C for three days followed by centrifugation at 13,000 rpm for 5 min. The bacterial precipitate was collected, sonicated and resuspended in PBS(10 mM, pH7.4), and 1 mL of the supernatant was mixed with 1 mL of 1% carboxymethyl cellulose (CMC, dissolved in PBS) and incubated at 37°C for 30 min with shaking (120 rpm). 1 mL of DNS reagent was added and the mixture was boiled for 5 min before absorbance was measured at 540 nm. A unit of enzyme activity was defined as per µmol of reducing sugar produced per minute. Total protein concentration was determined according to the Bradford method. A standard curve was drawn based on the different concentrations of bovine serum albumin (BSA) performed. The specific activity of cellulase was calculated by expressing the final product concentration (μmol reducing sugar/min) as units divided by the total protein (mg) of the sample. The control group was the experiment performed using untransformed E. coli Rosetta. The activity of the control group was 0.5 U/mg, which may have been the background noise of the experiment. Under our experimental conditions (37 ° C, pH 7.4), the specific activity of the crude enzyme solution of Bgls was 11.45 U/mg. This means that each milligram of the crude enzyme solution is capable of releasing 12 micromoles of reducing sugar per minute. This result confirmed the cellulase activity in our engineered strain and provided strong support for the co-application of cellulase and alginase.

2. Optimal reaction temperature of cellulase Bgls

To determine the optimal reaction temperature for cellulase, we mixed the crude enzyme solution and 1% CMC solution and determined the reducing sugar concentration using the 3, 5-dinitrosalicylic acid (DNS) method after incubation at 25 ° C, 37 ° C, and 55 ° C for 30 min.

3. Optimal reaction pH of cellulase Bgls

To determine the optimal reaction temperature for cellulase, we mixed the crude enzyme solution and 1% CMC solution at 37 ° C. After incubation at pH 5.8, 6.5, and 7.4 (PBS buffer) for 30 min, the concentration of reducing sugar was determined using the 3, 5-dinitrosalicylic acid (DNS) method. The optimum pH of Bgls was 6.5.

Experimental procedure:

Wash the kelp thoroughly using purified water to remove any accumulated dirt and dry it at 65°C for 2 hours. Soak 1g of dry kelp in water for 2 hours and cut into 1cm2 pieces. Crude enzyme solutions of cellulase and alginase were prepared using Tris-HCI (pH 7.4). 1g of dried kelp was mixed with 20 mL of the reaction system (10 mM Tris-HCI buffer, 10 mL of alginase solution, and different volumes of cellulase solution). After 24 hours of incubation at 37°C at 180 rpm. Samples were taken, kelp was filtered and washed, and then dried at 65°C to constant weight. The degradation rate was calculated by comparing the kelp weight before and after the treatment. All experiments were performed in triplicate and data will be expressed as mean plus SD.

Experimental procedure:

The recombinant plasmid was transformed into E. coli Rosetta competent cells. Positive transformants were screened on LB AGAR plates supplemented with ampicillin. Individual colonies were inoculated into LB broth and cultured with various concentrations of arabinose to induce SRRz expression. Bacterial growth was monitored until 28 h to assess the inhibitory effect of SRRz expression. All experiments were repeated three times, and data are presented as mean ± standard deviation (SD).