A genetically engineered bacterium with high self-aggregation ability and its construction method and application

Abstract

The invention discloses a genetically engineered bacterium with high self-aggregation ability and a construction method and application thereof, belonging to the field of genetic engineering technology. The genetically engineered bacterium is prepared by inactivating the gdpP gene of Lactobacillus plantarum WCFS1 and then transforming it. The nucleotide sequence of the gdpP gene after inactivation is shown in SEQ ID NO.1. The upstream and downstream homologous arm sequences and lox site sequences of the gdpP gene are amplified, the fusion fragments are obtained by overlapping PCR, the linear vector fragments are obtained by reverse PCR, the gdpP knockout vector is constructed by seamless cloning, the vector plasmid is electrotransformed, the gdpP deletion mutant is screened and verified, and the genetically engineered bacterium is constructed. The genetically engineered bacterium of the present application can enhance the aggregation ability of Lactobacillus plantarum and improve their potential as intestinal adhesion and colonization active carriers to deliver probiotics and functional components.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a genetic engineering bacterium with high self-aggregation ability and a construction method and application thereof. Background Art

[0002] Lactobacillus plantarum is a Gram-positive bacterium that occupies a wide range of ecological niches and can metabolize carbohydrates to produce lactic acid. It can not only change the flavor and texture of food and delay the spoilage of food, but also has good probiotic properties and therapeutic effects. Therefore, it is widely used in various fields related to food processing and health care. In order to better survive, reproduce and function, Lactobacillus plantarum needs to have excellent aggregation ability to resist stress and colonize the intestine. Lactobacillus plantarum can promote nutrient exchange and provide stress protection by forming multicellular aggregates, thereby creating a new ecological niche for itself that is conducive to survival and colonization. However, there is an upper limit to the aggregation ability of natural Lactobacillus plantarum. Therefore, it is necessary to introduce gene editing technology to further explore their potential as active carriers for intestinal adhesion and colonization, and to open up their application prospects in food processing and medical engineering.

[0003] gdpP is the gene encoding the second messenger molecule c-di-AMP degrading enzyme. Its deletion will cause an imbalance in the homeostasis of c-di-AMP in bacteria, thereby changing the aggregation characteristics of bacteria. As a Gram-positive bacterium, Lactobacillus plantarum is difficult to genetically manipulate. Currently, there is no report on the method of knocking out the gdpP gene through genetic engineering technology to improve the aggregation ability of Lactobacillus plantarum. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present application provides a genetically engineered bacterium with high self-aggregation ability and a construction method and application thereof.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] In a first aspect, the present application provides a genetically engineered bacterium with high self-aggregation ability, which is obtained by inactivating the gdpP gene of Lactobacillus plantarum WCFS1 and then modifying it. The nucleotide sequence of the modified gdpP gene after inactivation is shown in SEQ ID NO.1.

[0007] In one embodiment of the present application, the nucleotide sequence of the gdpP gene is shown as SEQ ID NO.2.

[0008] In a second aspect, the present application provides a method for constructing a genetically engineered bacterium with high self-aggregation ability, comprising the following steps:

[0009] S1. Extract genomic DNA of Lactobacillus plantarum WCFS1 and use the extracted genomic DNA as a template to amplify the upstream and downstream homology arm fragments of gdpP using primers gdpP-UF / gdpP-UR and gdpP-DF / gdpP-DR respectively;

[0010] S2, using the pNZ5319 vector as a template, using primers IF-gdpP-F1 and IF-gdpP-R1 to amplify the fragment containing the lox site;

[0011] S3, using the upstream and downstream homology arm fragments of gdpP and the fragment containing lox sites as templates, using primers IF-gdpP-F2 and IF-gdpP-R2 to perform overlapping PCR amplification to obtain a fusion fragment;

[0012] S4, using the pNZ5319 vector as a template, reverse PCR amplification was performed using primers pNZ5319-F and pNZ5319-R to obtain a PNZ5319 linear vector fragment;

[0013] S5. Connect the fusion fragment to the PNZ5319 linear vector fragment by seamless cloning technology to construct the knockout vector pNZ5319-UDgdpP;

[0014] S6, electro-transform the knockout vector pNZ5319-UDgdpP into WCFS1 competent cells, and obtain the target recombinants through screening;

[0015] S7. Extract the genomic DNA of the target recombinant and use its genomic DNA as a template to verify the knockout of the gdpP gene using primers V-gdpP-UF / V-gdpP-UR and V-gdpP-DF / V-gdpP-DR, respectively.

[0016] In one embodiment of the present application, the sequences of primers gdpP-UF / gdpP-UR and gdpP-DF / gdpP-DR are:

[0017] gdpP-UF: 5′-CGTCAATTCACGAATCAAAATGGGG-3′;

[0018] gdpP-UR: 5′-CGAAAAGATCTTTTTCATATTAAAATCCTCA-3′;

[0019] gdpP-DF: 5′-GAATCTAATTAGGAGTGAGTCTAAATG-3′;

[0020] gdpP-DR: 5′-CACATCTGAAATCGACTTAAACC-3′.

[0021] In one embodiment of the present application, the sequences of primers IF-gdpP-F1 and IF-gdpP-R1 are:

[0022] IF-gdpP-F1: 5′-GAAAAAGATCTTTTCGTACCGTTCGTATAATG-3′;

[0023] IF-gdpP-R1: 5′-CTCCTAATTAGATTCTACCGTTCGTATAGCATAC-3′.

[0024] In one embodiment of the present application, the sequences of primers IF-gdpP-F2 and IF-gdpP-R2 are:

[0025] IF-gdpP-F2: 5′-GATAACGCGCTCGAGCGTCAATTCACGAATC-3′;

[0026] IF-gdpP-R2: 5′-GTCAGCTTTAGAGATCTCACATCTGAAATCG-3′.

[0027] In one embodiment of the present application, the sequences of primers pNZ5319-F and pNZ5319-R are:

[0028] pNZ5319-F: 5′-AGATCTCTAAAGCTGACGGGGTAAAC-3′;

[0029] pNZ5319-R: 5′-CTCGAGCGCGTTATCGGTCCTTTAAT-3′.

[0030] In one embodiment of the present application, the amplification system, with a total volume of 50 μL, includes 25 μL 2×TaqPCR Mix; 1 μL template; 2 μL upstream primer; 2 μL downstream primer and 20 μL water.

[0031] In one embodiment of the present application, the amplification reaction procedure is: 95°C for 5 min; 95°C for 35 s, 55°C for 35 s, 72°C for 1 min, 30 cycles; 72°C for 10 min.

[0032] In one embodiment of the present application, the voltage of electroporation is 1.8-2.2 kV, and the time of electroporation is 4-5 ms.

[0033] In one embodiment of the present application, the target recombinant has chloramphenicol resistance but not erythromycin resistance, the concentration of chloramphenicol in the resistance plate is 10 μg / mL, and the concentration of erythromycin in the resistance plate is 30 μg / mL.

[0034] In one embodiment of the present application, the method for screening the target recombinant is: resuscitating the bacterial solution, streaking on a chloramphenicol plate, and culturing anaerobically at 37° C. The next day, a single colony is picked and spotted on a chloramphenicol and erythromycin resistance plate, respectively, and anaerobically cultured at 37° C. for 1-2 days to screen out the target recombinant with chloramphenicol resistance but not erythromycin resistance.

[0035] In one embodiment of the present application, the sequences of primers V-gdpP-UF / V-gdpP-UR and V-gdpP-DF / V-gdpP-DR are:

[0036] V-gdpP-UF: 5′-CGTTAACTTAACAGCAACTGACGATG-3′;

[0037] V-gdpP-UR: 5′-CTGTGGTTATACTAAAAGTCGTTTGTTGG-3′;

[0038] V-gdpP-DF: 5′-GCAGGATTGTTTATGAACTCTATTCAGG-3′;

[0039] V-gdpP-DR: 5′-CAAACTGAAAATGGCCACTGTCG-3′.

[0040] In a third aspect, the present application provides an application of a genetically engineered bacterium with high self-aggregation ability in the preparation of a product that promotes the adhesion and colonization of Lactobacillus plantarum.

[0041] Compared with the prior art, the present application constructs genetically engineered bacteria by amplifying upstream and downstream homologous arm sequences and lox site sequences of the gdpP gene, obtaining fusion fragments by overlapping PCR, obtaining linear vector fragments by reverse PCR, seamlessly cloning to construct a gdpP knockout vector, electroporating the vector plasmid, screening gdpP deletion mutants and verifying the knockout of the gdpP gene. The genetically engineered bacteria of the present application can enhance the aggregation ability of Lactobacillus plantarum and improve their potential to be used as intestinal adhesion and colonization active carriers to deliver probiotics and functional ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram showing the detection results of PCR amplification of the upstream and downstream homology arm fragments of gdpP in Example 1 of the present invention;

[0043] Figure 2This is the plasmid map of the knockout vector pNZ5319-UDgdpP constructed in Example 1 of the present invention;

[0044] Figure 3 This is a diagram showing the verification results of knocking out the gdpP gene of Lactobacillus plantarum in Example 2 of the present invention;

[0045] Figure 4 It is a line graph comparing the auto-aggregation rates of the gdpP knockout strain and the WCFS1 wild-type strain in Example 3 of the present invention;

[0046] Figure 5 This is an analysis diagram of bacterial aggregation of the gdpP knockout strain and the WCFS1 wild-type strain under a scanning electron microscope in Example 4 of the present invention. DETAILED DESCRIPTION

[0047] The following will provide a clear and complete description of the technical solutions in the embodiments of the present application in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0048] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range.

[0049] Unless otherwise specified, the percentages between liquids and liquids in the present invention are volume / volume percentages; the percentages between liquids and solids in the present invention are volume / weight percentages; the percentages between solids and liquids in the present invention are weight / volume percentages; and the rest are weight / weight percentages.

[0050] The present invention is further described below in conjunction with specific examples. Molecular biology experimental methods not specifically described in the following examples can be performed with reference to the methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kits and product instructions.

[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources, such as Lactobacillus plantarum WCFS1 (ATCC BAA-793), which is derived from ATCC (American type culture collection).

[0052] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0053] Example 1 Construction of knockout vector pNZ5319-UDgdpP

[0054] (1) PCR amplification fragment and vector

[0055] Using the genomic DNA of Lactobacillus plantarum WCFS1 as a template, PCR amplification was performed with primers gdpP-UF / gdpP-UR and gdpP-DF / gdpP-DR, and the upstream homology arm fragment U-gdpP and the downstream homology arm fragment D-gdpP of gdpP were cleanly recovered.

[0056] gdpP-UF: 5′-CGTCAATTCACGAATCAAAATGGGG-3′;

[0057] gdpP-UR: 5′-CGAAAAGATCTTTTTCATATTAAAATCCTCA-3′;

[0058] gdpP-DF: 5′-GAATCTAATTAGGAGTGAGTCTAAATG-3′;

[0059] gdpP-DR: 5′-CACATCTGAAATCGACTTAAACC-3′.

[0060] Using the pNZ5319 vector as a template, PCR amplification was performed using primers IF-gdpP-F1 and IF-gdpP-R1, and the fragment lox66-cat-lox71 containing the lox site was cleanly recovered.

[0061] IF-gdpP-F1: 5′-GAAAAAGATCTTTTCGTACCGTTCGTATAATG-3′;

[0062] IF-gdpP-R1: 5′-CTCCTAATTAGATTCTACCGTTCGTATAGCATAC-3′.

[0063] The above three fragments were used as templates, and overlapping PCR amplification was performed with primers IF-gdpP-F2 and IF-gdpP-R2. The single-band fusion fragment U-cat-D was obtained by gel excision and recovery.

[0064] IF-gdpP-F2: 5′-GATAACGCGCTCGAGCGTCAATTCACGAATC-3′;

[0065] IF-gdpP-R2: 5′-GTCAGCTTTAGAGATCTCACATCTGAAATCG-3′.

[0066] Using pNZ5319 vector as template, reverse PCR amplification was performed with primers pNZ5319-F and pNZ5319-R, and the linear vector fragment of PNZ5319 was obtained by gel excision and recovery.

[0067] pNZ5319-F: 5′-AGATCTCTAAAGCTGACGGGGTAAAC-3′;

[0068] pNZ5319-R: 5′-CTCGAGCGCGTTATCGGTCCTTTAAT-3′.

[0069] The reaction system and reaction procedure of the above PCR amplification are shown in Table 1 and Table 2.

[0070] Table 1 PCR reaction system

[0071]

[0072] Table 2 PCR reaction program

[0073]

[0074] (2) Seamless cloning technology connects fragments and vectors

[0075] Add 1.5 μL of linear vector fragment, 3.5 μL of fusion fragment U-cat-D and 5 μL of 2×Hieff Clone Enzyme Premix to a 200 μL centrifuge tube. Mix by pipetting, centrifuge at low speed and place at 50°C for 20 min. Remove the centrifuge tube and place on ice for 5 min before transformation.

[0076] Example 2 Knockout experiment of Lactobacillus plantarum gdpP gene

[0077] (1) Preparation of competent cells of Lactobacillus plantarum WCFS1

[0078] Streak, pick a single colony and inoculate it into 2 mL MRS medium, and culture it at 37°C for 12 h. Transfer 10 μL of the bacterial solution into 3 mL MRS medium supplemented with 1% glycine, and culture it at 37°C for 12 h. Inoculate this bacterial solution into 50 mL MRS medium supplemented with 1% glycine at a 2% inoculum, and culture it at 37°C until the OD 600≈0.6. Ice bath for 10 minutes, centrifuge at 4℃ for 10 minutes, discard the supernatant, and try not to leave any residue. Resuspend the precipitate with 0.5-1 times the volume of the original bacterial solution in ice-cold 30% PEG-1500. Ice bath again for 10 minutes, centrifuge at 4℃ for 10 minutes, and discard the supernatant. Finally, resuspend the precipitate with 1mL of pre-cooled 30% PEG-1500, dispense 50μL per tube into pre-cooled 1.5mL centrifuge tubes, and store in a -80℃ refrigerator.

[0079] (2) Electroporation of knockout vector pNZ5319-UDgdpP

[0080] After rinsing the electroporation cup with ultrapure water and anhydrous ethanol, blow it under ultraviolet light until there is no ethanol residue. Place the plasmid pNZ5319-UDgdpP, WCFS1 competent cells, and electroporation cup in an ice bath for 10 minutes, add 4-6 μL of the plasmid DNA to be transformed to 50 μL of competent cells, tap the tube wall intermittently to mix it, and place it in an ice bath for 10 minutes. Add the mixed solution to the pre-cooled 2mm electroporation transformation cup, tap the electroporation cup to ensure that the mixed solution is at the bottom of the electroporation cup and no foam is generated. Wipe off the condensed water and mist outside the electroporation tank, put the electroporation cup into the electroporator, and adjust the electroporator so that the voltage is 2 kV. After the electric pulse ends, quickly remove the electroporation cup from the electroporation tank and add 1 mL of pre-cooled MRS medium (added with 17.10% sucrose and 2.03% MgCl 2 ) and resuscitated at 37℃ for 2h.

[0081] (3) Screening of gdpP knockout strains

[0082] The resuscitation solution was streaked on a chloramphenicol plate and cultured anaerobically at 37°C. The next day, single colonies were picked and spotted on chloramphenicol and erythromycin resistance plates (chloramphenicol and erythromycin concentrations were 10 μg / mL and 30 μg / mL, respectively), and cultured anaerobically at 37°C for 1-2 days to screen out target recombinants with chloramphenicol resistance but not erythromycin resistance. The genomic DNA of the target recombinants was extracted and their DNA was used as a template to verify the knockout of the gdpP gene using primers V-gdpP-UF / V-gdpP-UR and V-gdpP-DF / V-gdpP-DR, respectively.

[0083] V-gdpP-UF: 5′-CGTTAACTTAACAGCAACTGACGATG-3′;

[0084] V-gdpP-UR: 5′-CTGTGGTTATACTAAAAGTCGTTTGTTGG-3′;

[0085] V-gdpP-DF: 5′-GCAGGATTGTTTATGAACTCTATTCAGG-3′;

[0086] V-gdpP-DR: 5′-CAAACTGAAAATGGCCACTGTCG-3′.

[0087] The reaction system and reaction procedure of PCR amplification are the same as those in Table 1 and Table 2.

[0088] Example 3 Comparison of the autoaggregation rates of the gdpP knockout strain and the WCFS1 wild-type strain

[0089] The gdpP knockout strain and the WCFS1 wild-type strain were cultured at the same initial concentration (OD 600 ≈0.01) were inoculated into MRS medium and cultured at 37°C for 16 h. 10 mL of bacterial solution was collected in sterile centrifuge tubes and centrifuged to remove the supernatant. The cell pellets in each sample were resuspended in PBS buffer and their OD 600 The value was uniformly adjusted to 0.6. The cell suspension was placed at 37°C for 12 hours, and 200 μL of liquid was taken from the top of the suspension every 2 hours. The absorbance of the supernatant was measured at 600 nm using a spectrophotometer to calculate the autoaggregation rate of each sample at the corresponding time point.

[0090] Example 4 Scanning electron microscopy observation of bacterial aggregation of the gdpP knockout strain and the WCFS1 wild-type strain

[0091] The gdpP knockout strain and the WCFS1 wild-type strain were cultured at the same initial concentration (OD 600 ≈0.01) were inoculated in MRS medium and cultured at 37°C for 24 hours. The bacterial samples were collected every 8 hours. The samples were fixed with 2.5% glutaraldehyde at 4°C for 12 hours and washed three times with PBS buffer. The washed samples were sequentially dehydrated with ethanol gradient, coated and freeze-dried, and finally imaged using a scanning electron microscope.

[0092] The present application is described in detail above in conjunction with specific implementation methods and exemplary embodiments, but these descriptions cannot be understood as limiting the present application. Those skilled in the art understand that, without departing from the spirit and scope of the present application, the technical solution of the present application and its implementation methods may be subjected to a variety of equivalent substitutions, modifications or improvements, all of which fall within the scope of the present application.

Claims

1. A genetically engineered bacterium with high self-aggregation ability, characterized in that: The genetically engineered bacteria is prepared by inactivating the gdpP gene of Lactobacillus plantarum WCFS1 and then transforming it. The nucleotide sequence of the transformed gdpP gene after inactivation is shown in SEQ ID NO.

1.

2. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 1, characterized in that: The following steps are involved: S1. Extract genomic DNA of Lactobacillus plantarum WCFS1 and use the extracted genomic DNA as a template to amplify the upstream and downstream homology arm fragments of gdpP using primers gdpP-UF / gdpP-UR and gdpP-DF / gdpP-DR respectively; S2, using the pNZ5319 vector as a template, using primers IF-gdpP-F1 and IF-gdpP-R1 to amplify the fragment containing the lox site; S3, using the upstream and downstream homology arm fragments of gdpP and the fragment containing lox sites as templates, using primers IF-gdpP-F2 and IF-gdpP-R2 to perform overlapping PCR amplification to obtain a fusion fragment; S4, using the pNZ5319 vector as a template, reverse PCR amplification was performed using primers pNZ5319-F and pNZ5319-R to obtain a PNZ5319 linear vector fragment; S5. Connect the fusion fragment with the PNZ5319 linear vector fragment by seamless cloning technology to construct the knockout vector pNZ5319-UDgdpP; S6. The knockout vector pNZ5319-UDgdpP was electroporated into WCFS1 competent cells, and the target recombinants were obtained by screening; S7. Extract the genomic DNA of the target recombinant and use its genomic DNA as a template to verify the knockout of the gdpP gene using primers V-gdpP-UF / V-gdpP-UR and V-gdpP-DF / V-gdpP-DR, respectively.

3. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The sequences of the primers gdpP-UF / gdpP-UR and gdpP-DF / gdpP-DR are: gdpP-UF: 5′-CGTCAATTCACGAATCAAAATGGGG-3′; gdpP-UR: 5′-CGAAAAGATCTTTTTCATATTAAAATCCTCA-3′; gdpP-DF: 5′-GAATCTAATTAGGAGTGAGTCTAAATG-3′; gdpP-DR: 5′-CACATCTGAAATCGACTTAAACC-3′.

4. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The sequences of the primers IF-gdpP-F1 and IF-gdpP-R1 are: IF-gdpP-F1: 5′-GAAAAAGATCTTTTCGTACCGTTCGTATAATG-3′; IF-gdpP-R1: 5′-CTCCTAATTAGATTCTACCGTTCGTATAGCATAC-3′.

5. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The sequences of the primers IF-gdpP-F2 and IF-gdpP-R2 are: IF-gdpP-F2: 5′-GATAACGCGCTCGAGCGTCAATTCACGAATC-3′; IF-gdpP-R2: 5′-GTCAGCTTTAGAGATCTCACATCTGAAATCG-3′.

6. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The sequences of the primers pNZ5319-F and pNZ5319-R are: pNZ5319-F: 5′-AGATCTCTAAAGCTGACGGGGTAAAC-3′; pNZ5319-R: 5′-CTCGAGCGCGTTATCGGTCCTTTAAT-3′.

7. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The voltage of the electroporation is 1.8-2.2 kV, and the time of the electroporation is 4-5 ms.

8. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The target recombinant has chloramphenicol resistance but not erythromycin resistance, the concentration of chloramphenicol in the resistance plate is 10 μg / mL, and the concentration of erythromycin in the resistance plate is 30 μg / mL.

9. The method for constructing a genetically engineered bacterium with high self-aggregation ability according to claim 2, characterized in that: The sequences of the primers V-gdpP-UF / V-gdpP-UR and V-gdpP-DF / V-gdpP-DR are: V-gdpP-UF: 5′-CGTTAACTTAACAGCAACTGACGATG-3′; V-gdpP-UR: 5′-CTGTGGTTATACTAAAAGTCGTTTGTTGG-3′; V-gdpP-DF: 5′-GCAGGATTGTTTATGAACTCTATTCAGG-3′; V-gdpP-DR: 5′-CAAACTGAAAATGGCCACTGTCG-3′.

10. Use of the method for constructing a genetically engineered bacterium with high self-aggregation ability according to any one of claims 2 to 9 in preparing a product promoting intestinal adhesion and colonization of Lactobacillus plantarum.

Images