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 and 1 tube of DH5α/ pheP gene. One tube of E. coli DH5α/ empty vector 2 and one tube of DH5α/ carrying PAL gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube orifice and plug, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the test tube orifice and plug 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. Plasmid extraction: Four kinds of plasmids (DH5α/ empty vector 1, DH5α/ empty vector 2, and 1 tube of DH5α/ carrying iaaH gene) were extracted according to the instruction of plasmid extraction kit. One tube of E. coli DH5α/iaaM, one tube of DH5α/ carrying galU gene, and one tube of DH5α/ carrying Arabinose promoter and mazF genes. The bacterial solution was centrifuged for 1min and then the precipitate was removed. 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.

2. Diluted primer

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) Target genes pheP, PAL and two kinds of vector DNA fragments were amplified by high fidelity enzyme Pfu. The reaction system is as follows:

The Pfu was 0.5 μL

10xPfu buffer 5 μL

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

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 TF, TR. The reaction system 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 plan:

Experimental procedure:

Engineered E. coli Rosetta strains expressing IaaM and IaaH and control strains were grown at 37 ° C in Luria-Bertani (LB) medium with shaking at 200 rpm. Because IAA was easily decomposed in light, the triangle flask was wrapped in tin foil and the shaker was shaded with newspaper to achieve light-sheltered conditions. Every 24 hours, 1.5 mL of the bacterial culture medium was taken, and 1 mL of the supernatant was collected after centrifugation (10,000 rpm, 1 min). For IAA assay, 1 mL of the supernatant was mixed with 1 mL of Salkowski reagent (2% of 0.5M FeCl₃, 35% perchloric acid) 1. 1 The mixture was incubated in the dark for 30 min, after which a pink color indicated the presence of IAA. The intensity of the color, which is proportional to the concentration of IAA, was measured by spectrophotometry at 530 nm. Standard curves were prepared with known concentrations of IAA for the quantification of IAA in bacterial cultures. All experiments were repeated three times, and data are presented as mean ± standard deviation (SD). Statistical significance between the engineered strain and the control strain was tested using Two-way ANOVA (two-way analysis of variance), with p < 0.05 considered statistically significant.

Experimental plan:

Experimental procedure:

The growth of bacteria was monitored by measuring OD600 at predetermined time points (4, 8, 16 h). At the same time, the cells were centrifuged, and the supernatant was used to quantify EPS by the anthrone sulfuric acid method. In brief, a sample of the supernatant was combined with an anthrone reagent, followed by the addition of concentrated sulfuric acid. The mixture was subsequently heated and the color development was measured by spectrophotometry at 620 nm. As the main component of EPS is polysaccharide, a standard curve of glucose was used to evaluate the EPS concentration in the sample. The EPS concentration was then normalized to OD600 to obtain EPS production per unit bacterial growth. All experiments were repeated three times, and data are presented as mean ± standard deviation (SD).

Experimental plan:

Experimental procedure:

1. We added arabinose to the bacterial solution to induce the expression of mRFP. The fluorescence intensity of mRFP was measured at 584 nm excitation and 607 nm emission using a microplate reader. This intensity was used as an indicator of arabinose promoter activity.

2. Fluorescence microscopy verification experiment:

(1) Sample preparation: appropriate amount (about 30 μl) of wild type Rosetta and Rosetta-engineered bacteria droplets were dropped onto the slide and covered with a cover slip.

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

(3) Insert the light stopper to interrupt the light path.

(4) Preheat for 5-10 min.

(5) Place the slide with the sample on the stage.

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

(7) Rotate the turntable of the spectroscope assembly to select the spectroscope assembly for observing mRFP (red fluorescence).

(8) Adjust the focal length through thick and thin screws.

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

Experimental procedure:

When the OD600 of the engineered and control strains was 0.6, 1 mM arabinose was added, and after 4 hours of induction, the fluorescence intensity was measured using a microplate reader and divided by OD600 to obtain the relative fluorescence intensity. In order to test the response of arabinose promoter to different concentrations of arabinose. When the OD600 of the engineered strain was 0.6, 0.1, 0.5, 1, 2, and 4 mM arabinose were added. The fluorescence intensity was measured after 4 hours of induction and divided by OD600 to obtain the relative fluorescence intensity.

mazF suicide gene was synthesized and cloned into the downstream of the arabinose promoter of pSB1A3 vector. The recombinant plasmid was transformed into E. coli Rosetta competent cells by the heat-shock method. Positive transformants were screened on LB AGAR plates supplemented with ampicillin.

Individual colonies were inoculated into LB broth and grown until the optical density, OD600, reached 0.6. Different concentrations of arabinose were added to the cultures to induce MazF expression. Bacterial growth was monitored at specific time intervals, namely 2, 6, and 14 h after induction, to assess the inhibitory effect of MazF expression. All experiments were repeated three times, and data are presented as mean ± standard deviation (SD).