This experiment page includes the protocol we used in the wet lab experiments, from obtaining the target fragment to build the plasmid, to transforming the constructed plasmid into P. putida, finally by systematic characterizaiton of the structure of manganese oxide, and the degradation performance of lignin.
Alternative CumA, source: Pseudomonas putida. On the NCBI website, the GenBank database was utilized to search for sequences. Click on "download datasets" to download gene sequences, protein sequences, and other information.
Primer5 was used to design primers.
1.3.1 Genome DNA was extracted from the cells
Lysozyme was added, and the sample was placed in a water bath for complete lysis (cell fragmentation). Nucleic acids were extracted by fragmentation, and impurities were removed. The resulting nucleic acids were purified.
1.3.2 PCR was used to amplify the known gene fragments
PCR amplification was performed using the PCR in vitro amplification technique with a PCR apparatus. A 0.2mL enzyme-free PCR reaction tube was used to add each reagent separately with a microsample gun in the order described below.
1.4.1 Gel electrophoresis of DNA
Rinse the gel mold and comb with distilled water, place them on the gel plate, and secure the comb. Prepare the gel. Once the gel has completely set at room temperature, carefully remove the comb and place the gel into the electrophoresis tank. Pour the electrophoresis buffer into the tank, ensuring the buffer level is 1mm below the gel surface. Remove any bubbles in the sample wells. After adding loading buffer to the DNA sample, slowly introduce the mixture into the submerged gel loading port with a pipette. Turn on the power, with red indicating the positive electrode and black the negative electrode. Ensure DNA samples migrate from the negative to the positive electrode (add one end close to the sample well). Typically, a voltage of 50V is used for electrophoresis, which can last for 90 minutes. Terminate electrophoresis based on the migration of the indicator dye. Post-electrophoresis, turn off the power, observe using nucleic acid dye staining and an ultraviolet detector, and record the electrophoresis results.
1.4.2 Recovery and purification of PCR products from gels
Under ultraviolet light, cut out agarose gel blocks containing the target DNA fragments. Weigh the gel block and add sol solution according to a specific ratio. Heat the sol in a 55℃ water bath, gently mixing until the gel is completely dissolved. Transfer the dissolved solution to an adsorption column and centrifuge to separate the DNA. Wash away impurities with a wash solution, and finally elute the DNA with the eluate to obtain purified PCR products. Store as instructed.
1.5.1 Double enzyme digestion of the target gene
Add the following reagents in proportion to a double digestion system in sterile centrifuge tubes.
1.5.2 Recovery and purification of restriction enzyme products
Estimate the relative amount of recovered target genes by AGAR gel electrophoresis and purification using a nucleic acid quantification apparatus.
1.6.1 Double restriction enzyme digestion of the expression vector
Add the following reagents in proportion to a double digestion system in sterile centrifuge tubes.
1.6.2 Recovery and purification of restriction enzyme products
Detect the digested vector by AGAR gel electrophoresis and recover and purify using a vector extraction kit.
Prepare the ligation system by adding the following reagents in proportion to a sterile centrifuge tube (the ratio of control expression vector to target gene should be 1:3-1:10; Ligate at 4℃ for 1 hour and store at 4℃).
1.8.1 Preparation of competent cells
Add single colonies to 5 mL LB liquid medium and incubate overnight at 37 ℃ at 180rpm with shaking. Inoculate 5% (2.5 mL) bacterial solution into a shaking flask containing 50mL LB and incubate at 37 ℃ for 2-3 hours to OD600 = 0.4-0.5. Place cultures in an ice water bath for 20 minutes. Collect cells by centrifugation at 5000rpm and 4℃ for 5 minutes. Discard the culture solution, trying to remove as much of it as possible. Suspend cells in 20 mL of 0.1 mol/L CaCl2 precooled in an ice bath for 20 minutes. Centrifuge at 5000 rpm for 5 minutes at 4℃ and decant the supernatant. Suspend cells again with 10mL of 0.1mol/L CaCl2 precooled, then centrifuge. Prepare competent cells by adding 2 mL of 0.1 mol/L CaCl2 precooled and gently suspending the cells. They can be used immediately or divided into 200 µL aliquots for future use and frozen at -80 ℃.
1.8.2 Transformation of plasmids
Transform the ligation products of the target gene and the expression vector into competent cells using an ice bath and heat shock method. Then, spread the cells on the surface of LB medium containing gentamicin and culture in an incubator at 30 ℃ for 12 hours. Set up a positive control group and a negative control group for comparative observation.
1.9.1 Enrichment cultivation
Transfer single colonies selected from the plates to 5 mL of liquid LB medium containing 1% gentamicin and incubate overnight at 30 ℃ at 200rpm.
1.9.2 Bacterial fluid was identified by PCR
Amplify the target gene fragment by PCR using bacterial broth as a template and verify the length of the target gene by agarose gel electrophoresis. The operating system is as follows:
1.10.1 Extraction of recombinant plasmids
Extract recombinant plasmids from Pseudomonas putida using a vector extraction kit. Digest the plasmids with single and double restriction enzymes, respectively. Preliminary identification of target genes is done by gel electrophoresis.
1.10.2 Sequence identification of recombinant plasmids
Once the above identification results are correct, send the recombinant plasmid to a company for sequence identification.
Carry out the scale-up culture in a clean bench. Store the obtained bacterial solution and transfer 5 μL of seed solution to 5 mL of LB liquid medium containing gentamicin, incubate overnight at 37 ℃, 220 rpm. Then, transfer to 50 mL of liquid LB medium at a volume ratio of 1:1000.
After the bacteria have been cultured for 6 hours, add 500 μL of IPTG to the medium to achieve a final concentration of 0.1 mM for induced expression. Subsequently, transfer the medium to a 30 ℃, 200 rpm shaker for 8 hours.
Remove the Erlenmeyer flask from the shaker and transfer the bacterial solution to a 50 mL centrifuge tube to collect the bacterial pellet, using centrifugation parameters of (4 ℃, 10000×g, 5 minutes). Wash the bacterial pellet three times with ddH2O, then resuspend the bacteria in 5 mL of PBS buffer. Subsequently, transfer the bacterial suspension to ice and disrupt the bacteria completely using a cell breaker with ultrasonication for 15 minutes (power 60%; Sonication for 3 seconds, intermittent for 3 seconds). Centrifuge the crushing solution at 10,000×g at 4 ℃ for 15 minutes, collect a small amount of supernatant and precipitate separately. Filter the remaining supernatant through a 0.22 μm ultrafiltration membrane and collect the filtrate for affinity chromatography.
2.4.1 Protein purification.
Equilibrate the column using 5 column volumes of equilibration buffer. Load the supernatant of the filtered lysate onto the balanced chromatography column, collecting approximately 1 mL of the lysate supernatant and labeling it for storage. Elute heteroproteins with 10 column volumes of equilibrium buffer, retaining approximately 1 mL of heteroprotein eluent for electrophoresis during the final column volume elution. Elute the target protein using 2 column volumes of elution buffer and collect all eluates. Wash the desalting column using 10 column volumes of 500 mM imidazole elution buffer. Clean the column using 10 column volumes of pure water. Seal the column with 5 column volumes of 20% ethanol and store tightly closed at 4~8 ℃.
2.4.2 Desalination
Clean the desalination column with 3 column volumes of pure water. Equilibrate the chromatography column with 5 column volumes of desalting buffer PB. Load 1.5 mL of the protein solution post-affinity chromatography onto the desalting column, retaining a small amount of eluted protein solution samples. Elute the protein of interest using 2 mL of desalting buffer and collect the protein of interest. Equilibrate the chromatography column with 5 column volumes of desalting buffer. Wash the desalination column using 10 column volumes of pure water. After sealing the column with 5 column volumes of 20% ethanol, seal and store at 4~8 ℃. Store all collected sample solutions at 4 ℃, adding glycerol to the samples to achieve a final concentration of 5% post-desalting and store at 4 ℃.
2.5.1 SDS polyacrylamide gel production
Utilize Solarbio's SDS-PAGE Gel Preparation Kit to prepare gels in the experiment. Leak detection: Identify the matching 1.5 mm rubber plate, rinse it well, dry it with lens polishing paper, and secure it on the dispenser. Conduct a leak test with deionized water. Preparation of separating gel: After 5 minutes, if the gel board does not leak, prepare the separating gel. Prepare 10 mL of 12% resolving gel according to the kit's ingredient table (Table 6). Filling and separating the gel: Pour out the water in the rubber board and absorb the water with filter paper. Take 5 mL of the mixed separating gel and gently pour it along the edge of the gel plate to prevent bubbles, and level the separating gel with water. Preparation of stacking gel. Prepare 5 mL of 5% stacking gel according to the kit's ingredient table (Table 7). Filling concentrated gel: A distinct horizontal line forms between the separating gel and the water, indicating that the separating gel has solidified. Pour out the water on the top layer of the separating gel, absorb the water with filter paper, take the mixed concentrated gel and gently pour it along the edge of the rubber plate, insert a comb with 11 wells, and let it stand for 30 minutes. Prepare running buffer (500 mL). Follow the ingredient table of the electrophoresis buffer (Table 8). Turn the gel maker upside down, ensuring the gel does not flow out. Load the solidified gel plate into the electrophoresis tank and add SDS-PAGE electrophoresis buffer to soak the gel for a period.
2.5.2 Sampling-sampling electrophoresis
Take 10 centrifuge tubes and label the samples as "pre-induction, post-induction, broken supernatant, broken precipitate, penetrating solution 1, penetrating solution 2, 5 μg pre-elution protein, 10 μg pre-eluting protein, 5 μg post-elution protein, 10 μg eluting protein" in the order of "1-1". Mix 40 μL of protein sample with 10 μL of 4× loading buffer in a 1.5 mL centrifuge tube, mix well, and heat in boiling water for 10 minutes. Allow to cool to room temperature. Load 10 μL of sample and 5 μL of protein marker (15-130 KDa). Initiate electrophoresis at a constant voltage of 80 V, increase to 120 V after the samples enter the separating gel, and stop electrophoresis when the blue bromophenol blue band migrates to about 1 cm from the lower end of the gel. Cut off the power, unplug and pour the electrode buffer into a large beaker. Unload: Remove the rubber plate from the electrophoresis instrument, pry the rubber plate open with a blade, and separate the concentrated rubber part of the gel. Staining: Add Coomassie Brilliant Blue to the extracted stacking gel for 15 minutes, then transfer to the decolorization solution for destaining. After the eluent no longer changes color, take a picture and store it.
Conduct the entire assay on ice. Prepare the assay system according to Table 9, prepare 4 samples for each system, and finally add 3 wells.
Set the microplate reader parameters: temperature 30 ℃, vibrating plate 30 seconds, wavelength 340 nm, kinetics 15 minutes, and 1-minute intervals, so we will have 15 data points for each well of the final sample.Based on the increase in absorbance at a specific wavelength and the molar extinction coefficient of the reaction product, calculate the amount of reaction product generated per minute, which can then be used to further calculate the enzyme activity and specific activity.
3.1.1 Obtain the standard name of the target gene or protein by referring to the literature.
3.1.2 Conduct a search on the NCBI website to obtain the input operon sequence.
Constitutive promoters enable gene expression to be activated in all tissues. Conduct a search on the NCBI website to obtain the constitutive promoter of the desired species.
3.3.1 Synthesis of sgRNA primers using the CRISPR-Cas system
Select the target DNA sequence: identify the gene to be edited and the purpose of editing, such as targeting a specific gene mutation, deletion, or replacement. Determine the target sequence: according to the editing purpose, determine the target sequence that needs to be edited in the target gene, usually a sequence of 20 nucleotides. Design the sgRNA sequence: sgRNA typically consists of two parts: the target sequence and the scaffold sequence. The scaffold sequences usually include transcription initiation sites at the 5' end and termination sites at the 3' end, which are critical for the proper functioning of the CRISPR-Cas9 system. The target sequence should be complementary to the target sequence of the target gene to ensure specificity. Determine the appropriate primer length: the primer length is usually 17-20 nucleotides, and the shorter the primer, the greater the likelihood of non-specific binding and shearing. The selection of length needs to be optimized according to different specific experiments and cell types. Determine the appropriate location of the sgRNA primer: usually, the appropriate location is selected in the promoter or exon region of the target gene. Use online tools for validation: Use online tools or software to verify the specificity and accuracy of sgRNA, such as Crispr design. Synthesis of sgRNA primers: The designed sgRNA sequence is synthesized into RNA molecules.
3.3.2 Construction of sgRNA-Cas9 plasmid and repair of DNA template plasmid
After preparing the sgRNA, the Cas9 protein and sgRNA need to be expressed. Methods such as gene transfection, electroporation, or viral vectors are usually used to introduce Cas9 and sgRNA into cells. These methods ensure that enough Cas9 and sgRNA reach the target gene for effective gene knockout.
3.3.3 The recombination of DNA sequences
Once Cas9 and sgRNA have successfully entered the cell, Cas9 binds to the sgRNA and cuts the DNA sequence of the target gene. Cas9 typically cuts DNA around a primer sequence specified in the sgRNA.
3.3.4 Verifying Successful typing
Purine screening, fluorescence detection, and PCR to detect the genome, RFP protein is knocked in.
Take the modified bacterial solution, dilute and spread on the resistant plate to form a monoclonal; use an autoclaved tip to pick a single colony and punch the tip into 5 mL containing... Shake the tube of resistant LB liquid medium and incubate it overnight at 37 ℃; take 1 μL of bacterial solution, add primers, ddH2O, Rapid TaQ, and Master Mix to configure the PCR reaction system, and adjust the PCR instrument program for PCR. Gel, electrophoresis buffer, sample loading, nucleic acid gel electrophoresis to detect PCR products.
PCR amplification of the DNA fragment to be tested; prepare 4 dNTPs and DNA polymerases containing DNA templates, primers, 4 radiolabeled dNTPs, add 4 ddNTPs in proportion, and adjust the instrument to start the reaction; The reaction products in four reaction tubes were separated by denaturing polyacrylamide gel electrophoresis, and the DNA sequence was estimated according to the length of the fragments and the terminal base information.
To verify the feasibility of the promoter, the transcription of the downstream ccmE gene was detected by QPCR. First, extract total bacterial RNA using an RNA extraction kit and reverse-transcribe the corresponding cDNA. Subsequently, add buffers, upstream and downstream primers, DNA polymerase, deoxyribonucleic acid, dyes, etc., sequentially, and obtain the amplification curve and dissolution curve using the SYBR-Green PCR instrument. Obtain the expression of the target gene by using the unmodified Pseudomonas putidatum as the control.
Seed the Pseudomonas aeruginosa seed solution into 5 mL of LB medium at an inoculum rate of 1% and activate overnight on a constant temperature shaker at 30℃ and 200 rpm.
Inoculate the activated seed solution into 50 mL of BL medium at a volume ratio of 1:1000 for expanded culture.
After 12 hours of further incubation, add 500 mg of manganese sulfate to 50 mL of BL medium as a manganese source to synthesize manganese oxides in co-culture with P. putida.
Collect organisms using a cryogenic high-speed centrifuge at 4000 rpm/min, then break the organisms using an ultrasonic crusher. Collect the precipitates after high-speed centrifugation and wash three times with ddH2O and DMF, followed by freeze-drying.
Characterize the resulting precipitates using SEM, XRD, XPS, IR, and other characterization tools.
POD-like enzyme activity test: Weigh the obtained manganese oxides and dissolve them by sonication using ddH2O at 2 mg/mL, and weigh 6 mg of TMB and dissolve it in 1 mL of DMSO to obtain a 25 mM solution of TMB. Add 10 μL of manganese oxides solution to 10 μL of acetate buffer pH 3.6 and TMB solution, and measure the UV absorbance at OD652nm. Laccase-like activity test.
Lac-like enzyme activity test: Add 970 μL of MES buffer (0.1 M, pH 6.8) sequentially with nanozymes (2 mg/mL), 10 μL of 2,4-dichlorophenol (2.5 mg/mL), 10 μL of 4-aminoantipyrine (100 μg/mL), followed by measurement of absorbance at 510 nm using Nanodrop one.
Use native maize stover as a lignocellulose model and add stover powder to ddH2O with the previously obtained manganese oxide nano-enzymes. Carry out the reaction on a magnetic stirring table at room temperature at a speed of 800 rpm.
5.1.1 Concentration change of lignocellulose
Take the supernatant before and after the reaction and measure the absorbance changes at OD280nm and OD355nm using Nanodrop.
5.1.2 Structural changes of lignocellulose
Take the lignin before and after the reaction and freeze-dry. Detect the changes in functional groups of the above samples using Fourier Infrared Spectroscopy.
5.1.3 Morphological changes of lignocellulose
Obtain the morphological changes of lignocellulose before and after the reaction using SEM electron microscopy.
Inoculate Pseudomonas aeruginosa into the medium for cultivation at a volume ratio of 1:1000. Set the medium as: de-glucose sugar M9 medium and lignin as carbon source M9 medium.
5.2.1 Changes in bacterial biomass
Incubate the above medium at 30 ℃, 220 rpm for 12 hours. After 12 hours, spread 100 μL of the culture solution on LB solid medium for incubation. After 12 hours, count the number of bacterial colonies.
5.2.2 Size of bacterial monoclonal colonies
Centrifuge the bacterial culture solution cultured to OD600=0.4, wash, and dilute with ddH2O to OD600=0.2. Then, aspirate 100 microliters of the bacterial solution and spread on solid desugared M9 medium and solid lignin desugared medium, respectively. Culture in an incubator at 30 oC for 48 hours. After 48 hours, photograph and sample the bacterial plates and record the differences in the size of the morphology of the monoclonal colonies.
5.2.3 Manganese oxides assist bacteria to utilize lignin
Transfer a certain number of bacteria to lignin M9 medium without glucose. Set up the following groups: (1) Deglycosylated lignin M9 medium; (2)Bacteria+deglycosylated lignin M9 medium; and (3) Bacteria+manganese oxides+deglycosylated lignin M9 medium. Detect the lignin changes after incubation in the incubator at 30 ℃, 220 rpm for 48 hours. The results are shown in the table below.