CN116463370A - Three-plasmid genome editing system for bacillus bailii HCK2 spore surface expression and construction and application thereof - Google Patents

Three-plasmid genome editing system for bacillus bailii HCK2 spore surface expression and construction and application thereof Download PDF

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CN116463370A
CN116463370A CN202211742436.9A CN202211742436A CN116463370A CN 116463370 A CN116463370 A CN 116463370A CN 202211742436 A CN202211742436 A CN 202211742436A CN 116463370 A CN116463370 A CN 116463370A
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bacillus
protein
expression
hck2
cas9
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罗楚平
朱桃
李彬
张双玉
王小花
田宝霞
罗科程
陈悦雯
朱晓文
谷晓红
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Huaiyin Institute of Technology
Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention discloses a three-plasmid genome editing system for bacillus beleiensis HCK2 spore surface expression, construction and application thereof, wherein the gene editing system comprises an editing vector pTN-Cas9 and any one or more of editing vectors pTK-GFP-CotB and pTC-RFP-CgeA, and the sequences of the editing vectors are respectively shown in SEQ ID NO. 1-3. The gene editing system can accurately and rapidly edit spore protein genes in bacillus bailii HCK2 genome, and can eliminate plasmids for continuous editing through high-temperature treatment, the editing system is adopted to realize the editing of bacillus bailii HCK2 genome and the collaborative co-expression of two fluorescent protein genes on the spore surface, and the construction of the gene editing system lays a foundation for developing novel biological products by using bacillus bailii spores as nano carriers.

Description

Three-plasmid genome editing system for bacillus bailii HCK2 spore surface expression and construction and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a multi-plasmid genome editing system for bacillus spore surface expression, and construction and application thereof.
Background
Bacillus spores are surrounded by an outer shell, which is a protein structure consisting of at least 20 polypeptides, a common outer shell protein being TasA, cgeA, cotB, cotG, etc. Spore expression has the following advantages: (1) The spore has no cell membrane structure, the fusion protein does not need to be subjected to membrane crossing, and the problems of inaccurate folding and positioning are effectively avoided; (2) The display range is wider, and the exogenous protein with larger molecular weight can be displayed; (3) The presence of ATP-dependent chaperones can aid in the correct folding of the fusion protein; (4) there is no apparent codon bias. The bacillus is used as a probiotic, has positive effect on intestinal health and stability, and the spore can be used as a novel mucosal vaccine delivery system. Although E.coli can also be used as an expression host for heterologous proteins, it cannot be used in reports of the production of edible vaccine vectors because it is only expressed intracellularly due to the presence of endotoxins. Bacillus beleiensis, a common food-safe beneficial microorganism, also has the advantage of secretory expression and spore surface expression, and thus has the potential to produce oral vaccines.
The gene editing system mainly edits tools: zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas systems based on artificial endonucleases. The CRISPR/Cas system, in particular to CRISPR/Cas9 technology, has wide application in prokaryotic or eukaryotic microorganisms, and has the advantages of simple design and operation, low cost, high efficiency, accuracy, wide universality and the like. Microorganisms may undergo multiplex cleavage of DNA under the influence of replication or intrinsic and extrinsic factors, so that the genome is unstable and the cell dies. At this point, the DNA in the cell will undergo repair, mainly Homology Directed Repair (HDR) and non-homologous end joining (NHEJ). The NHEJ pathway is only present in a very small number of bacteria such as bacillus and agrobacterium, and therefore, when editing the genome of a prokaryotic microorganism, it is necessary to artificially provide homologous recombination fragments of genes to be edited, and repair DNA fragmentation fragments by the HDR pathway to achieve deletion, mutation or insertion of genes. Principle of operation of CRISPR/Cas9 technology: the sheared target gene has a PAM region nearby, which can be identified by a complex with guiding function (sgRNA), and the sgRNA brings Cas9 protein to the region, so that the accurate knockout of the gene is realized.
Bacillus belicus HCK2 (Bacillus velezensis HCK), the species Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) belongs to the genus Bacillus and is a gram positive bacterium. During the growth process, bacillus encounters bad environments such as nutrient deficiency, metabolite accumulation and the like, and the bacillus forms spores through asymmetric division and resists high temperature, toxic chemicals, lyase and other factors causing cell damage. And secondary metabolite surface active substances, bacitracin, rocamycin, iturin and the like generated in the fermentation process of bacillus have antibacterial, antiviral and antitumor activities. The laboratory researches show that the antibacterial peptide produced by bacillus can inhibit the proliferation of the PDCoV virus and has the function of promoting the growth of cells. In recent years, spores of bacillus are used as carriers, and surface display of heterologous proteins has become a promising field of research. In 11 2001, istic successfully expressed the C-terminal fragment of Tetanus Toxin (TTFC) using the Bacillus subtilis exine CotB as a vector. The madder fuses the PEDV S protein COE1 antigen fragment and the bacillus subtilis capsid protein CotB, and constructs bacillus subtilis recombinant spore for cooperatively expressing the antigen through homologous crossing, so that the bacillus subtilis recombinant spore has better immunogenicity, promotes the growth of mice, and improves the number of cecum dominant bacteria.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a three-plasmid genome editing system for expressing the HCK2 spore surface of bacillus beleiensis, which is a multi-plasmid gene editing system for expressing CRISPR-Cas9 genes by adopting bacillus constitutive promoters, assisting fluorescent protein screening markers and bacillus temperature-sensitive replication origins. The system can simultaneously edit 2 genes of the genome of bacillus and continuously edit, realizes the synergistic expression of a plurality of heterologous proteins on the surface of the spore, improves the editing efficiency of the HCK2 gene of bacillus beleiensis, reduces the screening difficulty of positive transformants and promotes the development of the spore surface expression technology.
The invention also provides a construction method and application of the multi-plasmid genome editing system for bacillus spore surface expression.
The technical scheme is as follows: in order to achieve the above purpose, the multi-plasmid genome editing system for spore surface expression of bacillus beleiensis comprises an editing vector pTN-Cas9 and any one or more of editing vectors pTK-GFP-CotB and pTC-RFP-CgeA, wherein the sequences of the editing vectors are respectively shown in SEQ ID NO. 1-3.
Wherein, the editing vector pTN-Cas9 takes pTN as a basic vector to insert a Cas9 protein expression frame; the Cas9 protein expression frame comprises a p43 promoter, a gene for encoding the Cas9 protein and a t1t2 terminator, and the sequence SEQ ID NO.4 of the Cas9 protein expression frame is shown.
Wherein the editing vector pTK-GFP-CotB is based on pTK, and is inserted into an sgRNA1 expression frame, and encodes green fluorescent marker protein (GFP) and spore surface protein BvCotB genes; the sgRNA1 expression frame comprises a p43 promoter and a sgRNA1 sequence, and the sequence of the sgRNA1 expression frame is shown in SEQ ID NO. 5; the sequence of the green fluorescent marker protein (GFP) gene of the coding gene is shown in SEQ ID NO. 6; the sequence of the BvCotB gene for encoding the spore surface protein is shown in SEQ ID NO. 7.
Wherein the editing vector pTC-RFP-CgeA takes pTC as a basic vector, and is sequentially inserted into an sgRNA2 expression frame, a coded red fluorescent marker protein (RFP) and a spore surface protein BvCgeA; the sgRNA2 expression frame comprises a p43 promoter and a sgRNA2 sequence, and the sequence of the sgRNA2 expression frame is shown in SEQ ID NO. 8; the sequence of the coded red fluorescent marker protein (RFP) gene is shown in SEQ ID NO. 9; the sequence of the BvCgeA gene for encoding the spore surface protein is shown as SEQ ID NO. 10.
The construction method of the multi-plasmid genome editing system for bacillus bailii HCK2 spore surface expression comprises the following steps:
(1) Optimizing a Cas9 protein sequence according to bacillus codon preference, synthesizing a p43 promoter and a Cas9 protein coding gene to obtain a Cas9 protein expression frame, inserting the Cas9 protein expression frame into a pTN vector, and constructing an editing vector pTN-Cas9;
(2) Designing and synthesizing an sgRNA1 expression frame conforming to the codon preference of bacillus beliensis, taking spore surface protein BvCotB as a target site, and designing and synthesizing a homologous recombination fragment for spore surface expression of bacillus beliensis by using green fluorescent protein as a recombinant expression protein; sequentially carrying out enzyme digestion and enzyme ligation cloning on the designed and synthesized sgRNA1 expression frame and the homologous recombination fragment to a pTK vector multiple cloning site to obtain the gene editing vector pTK-GFP-CotB;
(3) Designing and synthesizing an sgRNA2 expression frame conforming to the codon preference of bacillus beleiensis; taking spore surface protein BvCgeA as a target site, designing and synthesizing a homologous recombination fragment for spore surface expression of bacillus bailii by taking red fluorescent protein as a recombination expression protein; and cloning the designed and synthesized sgRNA2 expression frame and the homologous recombination fragment to the pTC multi-cloning site sequentially through enzyme digestion and enzyme connection to obtain the gene editing vector pTC-RFP-CgeA.
The invention relates to an application of a multi-plasmid genome editing system for spore surface expression of bacillus bailii HCK2 in bacillus genome editing and spore surface expression of heterologous proteins or polypeptides.
Wherein, the specific steps of expressing the heterologous protein or polypeptide on the surface of the spore are as follows:
(1) Respectively transferring pTN-Cas9, pTK-GFP-CotB and pTC-RFP-CgeA into E.coli competent cells DH5a, and extracting transformants to obtain a large number of plasmids;
(2) Respectively combining pTN-Cas9 with pTK-GFP-CotB and pTC-RFP-CgeA plasmids in pairs, or jointly transferring three plasmids into the same bacillus HCK2; coating the culture medium on a solid flat culture medium after culturing;
(3) Firstly, observing the conversion plate to fluoresce under blue light, and then, carrying out PCR verification by extracting genome to screen recombinant bacteria;
(4) The recombinant bacteria are subjected to high temperature of 50 ℃ to eliminate plasmids;
(5) And (3) taking recombinant bacteria with eliminated plasmids as host bacteria, and repeating the steps (2) - (4) with pTN-Cas9 plasmids and a vector pTK-GFP-CotB or pTC-RFP-CgeA containing another target protein to obtain the recombinant bacillus capable of simultaneously expressing green and red fluorescent proteins.
The bacillus recombinant strain capable of simultaneously expressing a plurality of different exogenous proteins is a three-plasmid genome editing system which is introduced into bacillus bailii HCK 2.
Preferably, the recombinant bacillus beliae HCK2 constructed by introducing the three-plasmid genome editing system into the bacillus beliae HCK2 can express a plurality of different exogenous proteins at the same time, and lays a foundation for the preparation of multiple seedlings.
The invention relates to a CRISPR-Cas9 system-based multi-plasmid genome editing system for assisting in fluorescent protein screening markers and temperature-sensitive replication origins, which can be used for the synergistic heterologous expression of a plurality of heterologous proteins or polypeptides on the surface of bacillus beijerinus HCK2 spores, and realizes the replacement knock-in of fluorescent marker genes and target genes. The system adopts a temperature sensitive replication origin, and constructs pTN-Cas9 by fusing a Cas9 protein expression frame of a p43 promoter; by adopting a temperature-sensitive replication origin, fusing a p43 promoter, (green and red) fluorescent protein expression frame, target protein expression frames (CotB and CgeA) and sgRNA expression frames to construct pTK-GFP-CotB and pTC-RFP-CgeA, bacillus such as bacillus bailii HCK2 genome can be accurately, rapidly and continuously edited, and the co-expression of multiple genes on the spore surface of bacillus bailii is realized.
Specifically, the invention adopts the constitutive promoter p43, can strongly start the expression of Cas9 protein in the late logarithmic growth phase of bacillus such as Bei Lai bacillus HCK2, and further promotes the application of the CRISPR-Cas9 system in Bei Lai bacillus HCK2 gene editing. The invention constructs a Cas9 protein expression frame (named as P43-Cas 9) comprising a P43 promoter, and a gene for encoding the Cas9 protein and a t1t2 transcription terminator.
The invention designs and synthesizes sgRNA1 and sgRNA2 sequences according to the nucleic acid sequence of Bei Lai bacillus HCK2 spore surface protein BvCotB, bvCgeA. Fusing bacillus constitutive expression promoter p43 and designed sgRNA1 sequence by adopting an overlap PCR technology to construct a p43-sgRNA1 transcription frame; the overlapping PCR technology is adopted to fuse bacillus constitutive expression promoter p43 and designed sgRNA2 sequence to construct p43-sgRNA2 transcription frame.
Wherein, the homologous recombination fragments are designed and synthesized according to the nucleic acid sequence of the BvCotB encoding Bei Lai bacillus HCK2 spore surface protein and the BvGFP encoding green fluorescent protein; the homologous recombination fragments are designed and synthesized according to the nucleic acid sequence of the BvCgeA encoding the HCK2 spore surface protein of bacillus beidellii and the RFP encoding the red fluorescent protein.
In the invention, a gene editing system is established in bacillus belicus relatively to bacillus subtilis, bacillus licheniformis and bacillus polymyxa. In particular, bacillus bailii is less reported as a beneficial microorganism for surface expression by its spores. The invention adopts a temperature sensitive replication origin, constitutive expression optimization Cas9 protein, a designed sgRNA sequence and a spore protein homologous exchange frame of fusion fluorescent protein, and successfully constructs a three-plasmid gene editing system capable of successfully editing the DNA of bacillus bailii genome. Compared with the traditional single plasmid editing system and the double plasmid editing system which can only edit a single gene, the invention adopts a three-plasmid editing system, and can simultaneously edit 2 genes in bacillus bailii HCK 2. In addition, the gene editing system is adopted to realize the synergistic expression of 2 fluorescent proteins on the surface of the bacillus bailii HCK2 spore. The invention also adopts a temperature-sensitive replication origin, and three plasmids can be eliminated in the genetic engineering strain after successful editing, and then continuous editing can be carried out. At present, the spore surface expression of Bei Lai bacillus is rarely reported, and the research on spore proteins is also rarely carried out. According to the invention, a homologous comparison method is adopted, and according to the spore protein nucleic acid sequence of bacillus subtilis, the potential spore protein of Bei Lai bacillus HCK2 is discovered, and is used as a carrier, and fluorescent protein or antigen is connected through a 5Gly linker. The protein adopts Bei Lai bacillus HCK2 spore proteins BvCotB and BvCgeA and GFP and RFP, which lays a foundation for simultaneously displaying various exogenous proteins, especially antigens of viruses, and particularly provides a technical means for developing multi-linked oral vaccines.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
in contrast to bacillus subtilis, bacillus licheniformis and bacillus polymyxa, the present invention first established three plasmid gene edits in bacillus beleiensis. In particular, bacillus bailii is less reported as a beneficial microorganism for surface expression by its spores. The invention adopts a temperature sensitive replication origin, constitutive expression optimization Cas9 protein, a designed sgRNA sequence and a spore protein homologous exchange frame of fusion fluorescent protein, and successfully constructs a three-plasmid gene editing system capable of successfully editing the DNA of bacillus bailii genome. Compared with the traditional single plasmid editing system and the double plasmid editing system which can only edit a single gene, the invention adopts a three-plasmid editing system, and can simultaneously edit 2 genes in bacillus bailii HCK 2. In addition, the gene editing system is adopted to realize the synergistic expression of 2 fluorescent proteins on the surface of the bacillus bailii HCK2 spore. The invention also adopts a temperature-sensitive replication origin, and three plasmids can be eliminated in the genetic engineering strain after successful editing, and then continuous editing can be carried out.
After the Bei Lai bacillus is successfully edited, the plasmid can be eliminated at high temperature, and the resistance gene residue is avoided, so that the gene editing system has the characteristics of being green and environment-friendly; the characteristics of the food safety level of the original strain can be kept.
The Bei Lai bacillus HCK2 spore proteins BvCotB and BvCgeA are compared with the bacillus subtilis JCL16, have higher homology with corresponding spore proteins, are novel spore proteins, can be used as a carrier to express heterologous proteins, and most reports about the novel bacillus are bacillus subtilis, but few reports about Bei Lai bacillus are available.
The Bei Lai bacillus HCK2 used in the invention can produce bacitracin L, ubiquitin, surfactant and rocamycin, has antibacterial and antiviral effects, can be used as a carrier for expressing heterologous proteins to immunize animals, and can improve immunity.
The recombinant bacillus bailii HCK2 constructed by the invention can express a plurality of different exogenous proteins at the same time, and lays a foundation for the preparation of multiple seedlings.
Drawings
FIG. 1A is a pTN-Cas9 plasmid map; b is a pTN-Cas9 plasmid enzyme digestion electrophoresis chart: xhoI single cleavage 10513bp, xhoI/kpnI multiple cleavage 7781bp/2845bp;
FIG. 2A shows the construction process of pTK-GFP-CotB plasmid; b is pTK-GFP-CotB plasmid enzyme digestion electrophoresis pattern: xhoI single restriction enzyme is 8080bp, xhoI/BamHI multiple restriction enzyme is 5350bp/2730bp; c is a GFP recombinant bacterium conversion plate, and emits green light under blue light; d is GFP displayed by GFP recombinant bacteria under a laser confocal microscope: IF: recombining spores under fluorescence; merge: a coincidence diagram;
FIG. 3A shows the construction process of pTC-RFP-CgeA plasmid; b is an enzyme digestion electrophoresis diagram of pTC-RFP-CgeA plasmid, wherein the single enzyme digestion of XhoI is 8060bp, and the multiple enzyme digestion of XhoI/BamHI is 5350bp/2710bp; c is RFP recombinant bacterium conversion plate, and emits red light under blue light; d is RFP displayed under an RFP recombinant strain laser confocal microscope: IF: recombining spores under fluorescence; merge: a coincidence diagram;
FIG. 4A shows a GFP-RFP recombinant bacterium transformation plate, which is emitted under blue light; b is GFP-RFP displayed under a laser confocal microscope: IF1: recombining spores under green fluorescence; IF2: recombining spores under red fluorescence; merge: a coincidence diagram;
in FIG. 5, A is a homology comparison result of Bei Lai bacillus HCK2 spore protein CotB and bacillus subtilis JCL16 spore protein CotB, and the homology can reach 41.99%; b is Bei Lai bacillus HCK2 spore protein CgeA, and the homology can reach 62.24% when the B is the homology comparison result with bacillus subtilis JCL16 spore protein CgeA;
FIG. 6 is a schematic diagram showing the co-display of two fluorescent proteins in Bacillus Bei Lai HCK2 using a three-plasmid gene editing system.
Detailed Description
The invention is further described below with reference to the drawings and examples.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Bacillus bailii HCK2 used in the invention has a preservation number CCTCC NO: m2019396. The preservation is disclosed in Chinese patent 2019107786075, which is provided by Huaiyin institute of technology.
The Bacillus subtilis JCL16 (preservation number: CCTCC NO: M2018336) used in the present invention has been publicly preserved in the prior patent CN111411053A, and is provided by the national institute of technology for wild type strain as usual.
pTN, pTK, pTC the sequence of the original vector gene is shown in the application patent 'E.coli-bacillus shuttle plasmid vector, and the construction method and application thereof (application number 202111297649.0)'; which have respectively different resistance genes, which are neomycin resistance genes Neo in turn R Kanamycin resistance gene Kana R Chloramphenicol resistance gene Cl R The method comprises the steps of carrying out a first treatment on the surface of the The corresponding double or multiple antibody plates were used in example 5.
Example 1
The pTN-Cas9 plasmid was constructed by insertion of the Cas9 expression cassette into the pTN vector optimized by Bio Inc. (FIG. 1).
The Cas9 protein expression cassette comprises a p43 promoter, a Cas9 protein and a t1t2 terminator, and the sequence of the Cas9 protein expression cassette is shown as SEQ ID No. 4; the pTN-Cas9 editing vector has the sequence shown in SEQ ID NO. 1.
Example 2
This example relates generally to the construction of a pTK-GFP-CotB recombinant plasmid (FIGS. 2A and B) fused to the sgRNA1 expression cassette of the p43 promoter and to the BvCotB protein coding gene of the green fluorescent protein (FIG. 5A).
(1) The P43 promoter and the sgRNA1 gene fragment synthesized by biological company are used as templates, P43BAF2/P43BABR2 and COTBSGF2/ECORSGR2 are respectively used as primers, the P43 promoter and the sgRNA1 gene sequence are amplified, the two fragments are purified and recovered, an expression frame (the sequence of which is shown as SEQ ID NO. 5) of the P43-sgRNA1 is constructed through fusion by an overlapping PCR technology, and the expression frame is purified and recovered through BamHI/EcoRI double digestion and is connected with a double digested (BamHI/EcoRI) pTK vector at 4 ℃ overnight, and the expression frame is transformed into E.coli DH5a to extract plasmids, thus obtaining the pTK-sgRNA1 recombinant plasmid.
(2) Fusion of BvCotB protein and GFP protein by overlap PCR
The BvCotB protein gene fragment synthesized by biological company is used as a template, the sequence of the BvCotB protein gene fragment is shown as SEQ ID NO.7, and primers COTBUF2/COTBUR2 and COTBDF2/COTBDR2 are respectively used for amplifying the upstream and downstream of the BvCotB protein gene; amplifying the corresponding gene fragment (the sequence of which is shown as SEQ ID NO. 6) by using COBGFPF2/COBGFPR2 as a primer and GFP optimally synthesized by a company as a template; the upstream fragment of the purified CotB protein gene is overlapped with GFP product, the overlapped product is overlapped with the downstream fragment of the CotB protein gene, the complete overlapped gene fragment is digested with EcoR I/Xho I, and the double digested pTK-sgRNA1 recombinant plasmid (EcoR I/Xho I) is connected with the recombinant plasmid at 4 ℃ overnight, transformed into Escherichia coli DH5a, and the plasmid is extracted to obtain the pTK-GFP-CotB vector (figures 2A and B), the sequence of which is shown as SEQ ID NO.2.
Example 3
This example mainly relates to the construction method of pTC-RFP-CgeA recombinant plasmid fused with sgRNA2 expression cassette of p43 promoter and BvCgeA protein coding gene (FIG. 5B) of red fluorescent protein.
(1) The P43 promoter and the sgRNA2 gene fragment synthesized by biological company are used as templates, P43AF2/P43AR2 and CGEASGF2/CGEASGR2 are respectively used as primers, the P43 promoter and the sgRNA2 gene sequence are amplified, the two fragments are purified and recovered, an expression frame (the sequence of which is shown as SEQ ID NO. 8) of the P43-sgRNA2 is constructed through fusion by an overlapping PCR technology, and the expression frame is recovered through double digestion purification of BamH I/Xba I and is connected with a pTK vector subjected to double digestion (BamH I/Xba I) at 4 ℃ overnight, and is converted into escherichia coli DH5a to extract plasmids, thus obtaining the pTC-sgRNA2 recombinant plasmids.
(2) Fusion of the protein BvCgeA with the RFP protein by overlap PCR
The BvCgeA protein gene fragment synthesized by biological company is used as a template (the sequence of the BvCgeA protein gene fragment is shown as SEQ ID NO. 10), and primers CGEAUF2/CGEAUR2 and CGEADF2/CGEADR2 are used for expanding the upstream and downstream of the CgeA protein gene respectively; amplifying corresponding gene fragments (the sequence of which is shown as SEQ ID NO. 9) by using CGEARFPF2/CGEARFPR2 as a primer and using RFP optimally synthesized by a company as a template; the upstream fragment of the purified BvCgeA protein gene is overlapped with the RFP product, the overlapped product is overlapped with the downstream fragment of the BvCgeA protein gene, the complete overlapped fragment is subjected to multi-enzyme digestion by EcoR I/Xho I, and is connected with a pTK-sgRNA2 recombinant plasmid subjected to multi-enzyme digestion (Xba I/Xho I) at 4 ℃ overnight, and the recombinant plasmid is transformed into escherichia coli DH5a to obtain a pTK-RFP-CgeA vector (figures 3A and B), wherein the sequence of the pTK-RFP-CgeA vector is shown as SEQ ID NO.3.
The primer sequences used in the present invention are shown in Table 1.
TABLE 1 primer sequences
Note that: the sites of the introduced cleavage are underlined.
Example 4
This example relates generally to the propagation and preparation of recombinant plasmids in E.coli.
Three tubes of 100ul of E.coli competent cells DH5a were taken from a-80℃refrigerator and rapidly placed in ice;
2uL of the carriers in the embodiment 1, the embodiment 2 and the embodiment 3 are respectively added, lightly mixed, placed in ice water bath for 30min, placed in water bath at 42 ℃ for 45s, quickly transferred to ice for 2min, added with 900uL of LB culture medium, and shake-resuscitated and cultured for 1 to 1.5h at 37 ℃;
100uL of the transformation solution was pipetted and coated on the sample containing Amp r Reversely culturing the resistant LB solid medium for 12-16 hours at 37 ℃, and selecting single colony to amplify in 5mL LB medium; the transformant was extracted, subjected to restriction enzyme digestion, and subjected to sequencing verification by a company to obtain plasmids pTN-Cas9, pTK-GFP-CotB, pTC-RFP-CgeA.
Example 5
The present implementation effort mainly involved the realization of the synergistic display of two fluorescent proteins in Bei Lai bacillus HCK2 using a three-plasmid gene editing system (fig. 6).
1. A preparation method of bacillus beleiensis HCK2 competence.
The preparation method of the bacillus belicus HCK2 culture medium comprises the following steps:
the table shows GCHE medium, GE medium without casein, 500ml for example
Nutrient broth medium: the nutrient broth medium 19g was weighed in 1L of ultra pure water, 121℃and autoclaved for 20min.
The preparation method of bacillus beleidsi HCK2 competence comprises the following steps:
(1) A single colony of Bacillus bailii HCK2 was picked and inoculated in 5mL of antibiotic-free GCHE liquid medium for 12h at 37℃with shaking at 160 rpm/min.
(2) 1mL of Bacillus bailii HCK2 bacteria solution is added into 24mL of new antibiotic-free GCHE liquid culture medium, and the mixture is cultured on a shaking table at 28 ℃ under shaking at 180 rpm/min.
(3) 1mL of culture solution is respectively sucked in five time periods of 2h, 2.5h, 3h, 3.5h and 4h, and is added into 1mL of antibiotic-free GE liquid culture medium for 1h at 37 ℃ under shaking culture at 200 rpm/min.
(4) Collecting fungus thallus in five time periods after centrifugation at 5000r for 5min, re-suspending and precipitating with supernatant, packaging 100uL of each fraction, and preserving at-80deg.C; the competent of the conversion efficiency of about 3h is adopted in the follow-up.
2. The method for transferring the plasmid into competent bacillus beliae HCK2 comprises the following steps:
(1) Firstly, transferring 2uL of pTN-Cas9 and pTK-GFP-CotB plasmids into 100uL of bacillus bailii HCK2 competent cells in example 4, carrying out shaking culture at 37 ℃ and 100rpm/min for 0.5h, then carrying out shaking culture at 180rpm for 1.5h, adding bacterial liquid into a multi-antibody (neomycin/kanamycin, 10 ug/mL) nutrient broth culture medium at a volume ratio of 1:100, carrying out shaking at 180rpm, carrying out overnight culture at 37 ℃, finally, uniformly coating 100uL of bacterial liquid on LB resistant plates containing neomycin (10 mug/mL) and kanamycin (10 mug/mL), and carrying out overnight culture in a 37 ℃ incubator;
(2) By observing the transformation plate under blue light, a green fluorescent mutant strain can be observed, a single colony is selected on a multi-antibody (neomycin/kanamycin, 10ug/mL each) LB medium, a recombinant bacillus belius HCK2 genome is extracted, and then a correct strain is selected through PCR verification (figures 2C and D);
(3) Inoculating recombinant bacteria to antibiotic-free liquid LB, culturing at 50 ℃ at 200rpm for 48 hours, diluting and coating the bacterial liquid 10 times of the bacterial liquid on a polyclonal antibody (neomycin/kanamycin, 10ug/mL each) LB plate without antibiotic, and culturing at 50 ℃ overnight to obtain the recombinant bacteria with plasmid elimination;
(4) Preparing competent cells by taking the recombinant bacteria with the plasmids eliminated in the step (3) as host bacteria, transferring pTN-Cas9 and pTC-RFP-CgeA plasmids into the competent cells of recombinant bacillus bailii HCK2, and repeating the steps (1) - (3) under the corresponding multi-antibody to prepare bacillus strains capable of generating green and red simultaneously (figures 4A and B).
In the above experiments, 2uL of pTN-Cas9 and pTC-RFP-CgeA plasmids were transferred together into 100uL of Bacillus bailii HCK2 competent cells of example 4, and red fluorescent mutants were prepared according to steps (1) to (3) under the corresponding polyclonal antibody (FIGS. 3C and D).
In addition, plasmids pTN-Cas9 and pTK-GFP-CotB were transformed according to the methods described above; plasmids pTN-Cas9 and pTC-RFP-CgeA; or plasmids pTN-Cas9, pTK-GFP-CotB and pTC-RFP-CgeA; after the cultivation, the cells were plated on corresponding resistant plates and incubated overnight in an incubator at 37 ℃. The plate can observe green and red fluorescent single colonies under blue light, and respectively observe green and red bacillus berryis under a laser confocal microscope, which shows that the multi-plasmid gene editing system successfully operates, 2 genes can be edited simultaneously, and the efficiency of gene editing is shown in table 3.
TABLE 3 editing efficiency of plasmid Gene editing System for simultaneously editing 2 genes
The above-described redness alone or greenness is the result of double plasmid gene editing, while greenness and redness are the result of three plasmid gene co-editing. The invention mainly emphasizes that a gene editing system is constructed in bacillus belicus for the first time, and BvCotB and BvCgeA proteins are successfully utilized as carriers, so that two fluorescent proteins are cooperatively displayed. The success of the editing system and spore surface expression lays a foundation for the subsequent use of spore expression functional proteins of bacillus bailii. After the Bei Lai bacillus is successfully edited, the plasmid can be eliminated at high temperature, and the resistance gene residue is avoided, so that the gene editing system has the characteristics of being green and environment-friendly; the characteristics of the food safety level of the original strain can be kept.

Claims (8)

1. A three-plasmid genome editing system for bacillus bailii HCK2 spore surface expression is characterized by comprising an editing vector pTN-Cas9 and any one or more of editing vectors pTK-GFP-CotB and pTC-RFP-CgeA, wherein the sequences of the editing vectors are respectively shown as SEQ ID NO. 1-3.
2. The three plasmid genome editing system for bacillus belay HCK2 spore surface expression of claim 1, wherein the editing vector pTN-Cas9 is inserted with a Cas9 protein expression cassette based on pTN; the Cas9 protein expression frame comprises a p43 promoter, a Cas9 protein coding gene and a t1t2 terminator, and the sequence SEQ ID NO.4 of the Cas9 protein expression frame is shown.
3. The three-plasmid genome editing system for spore surface expression of bacillus beleiensis HCK2 according to claim 1, wherein the editing vector pTK-GFP-CotB is a pTK-based vector, inserted into a p43-sgRNA1 expression cassette, encoding a green fluorescent marker protein (GFP) and a spore surface protein BvCotB gene; the p43-sgRNA1 expression frame comprises a p43 promoter and a sgRNA1 sequence, and the sequence of the p43-sgRNA1 expression frame is shown as SEQ ID NO. 5; the sequence of the coded green fluorescent marker protein (GFP) gene is shown as SEQ ID NO. 6; the sequence of the BvCotB gene for encoding the spore surface protein is shown in SEQ ID NO. 7.
4. The three-plasmid genome editing system for spore surface expression of bacillus belay HCK2 according to claim 1, wherein the editing vector pTC-RFP-CgeA is a pTC-based vector, and p43-sgRNA2 expression cassette, red fluorescent marker protein (RFP) encoding and spore surface protein BvCgeA gene are sequentially inserted; the p43-sgRNA2 expression frame comprises a p43 promoter and a sgRNA2 sequence, and the sequence of the p43-sgRNA2 expression frame is shown in SEQ ID NO. 8; the sequence of the coded red fluorescent marker protein (RFP) gene is shown in SEQ ID NO. 9; the sequence of the BvCgeA gene for encoding the spore surface protein is shown as SEQ ID NO. 10.
5. A method of constructing a three plasmid genome editing system for surface expression of bacillus beleiensis HCK2 spores according to claim 1, preferably comprising the steps of:
(1) Optimizing a Cas9 protein sequence according to bacillus codon preference, synthesizing a p43 promoter and a Cas9 protein coding gene to obtain a Cas9 protein expression frame, inserting the Cas9 protein expression frame into a pTN vector, and constructing an editing vector pTN-Cas9;
(2) Designing and synthesizing an sgRNA1 expression frame conforming to the codon preference of bacillus beleiensis, taking spore surface protein BvCotB as a target site, and designing and synthesizing a homologous recombination fragment for expressing the spore surface of bacillus beleiensis HCK2 by using green fluorescent protein as a recombinant expression protein; sequentially carrying out enzyme digestion and enzyme ligation cloning on the designed and synthesized sgRNA1 expression frame and the homologous recombination fragment to a pTK vector multiple cloning site to obtain the gene editing vector pTK-GFP-CotB;
(3) Designing and synthesizing an sgRNA2 expression frame which accords with the codon preference of bacillus bailii 2; taking spore surface protein BvCgeA as a target site, designing and synthesizing a homologous recombination fragment for spore surface expression of bacillus bailii by taking red fluorescent protein as a recombination expression protein; and cloning the designed and synthesized sgRNA2 expression frame and the homologous recombination fragment to the pTC multi-cloning site sequentially through enzyme digestion and enzyme connection to obtain the gene editing vector pTC-RFP-CgeA.
6. Use of the three-plasmid genome editing system for spore surface expression of bacillus beleiensis HCK2 according to claim 1 for genome editing of bacillus beleiensis, spore surface expression of a heterologous protein or polypeptide.
7. The use according to claim 6, wherein the specific steps for expressing the heterologous protein or polypeptide on the spore surface are as follows:
(1) Respectively transferring pTN-Cas9, pTK-GFP-CotB and pTC-RFP-CgeA into E.coli competent cells DH5a, and extracting transformants to obtain a large number of plasmids;
(2) Binding pTN-Cas9 with any one of pTK-GFP-CotB and pTC-RFP-CgeA plasmids respectively to transform into bacillus HCK2 competence; after culturing, coating the culture medium on a resistance flat plate culture medium;
(3) Firstly, observing the conversion plate to fluoresce under blue light, and then, carrying out PCR verification by extracting genome to screen recombinant bacteria;
(4) The recombinant bacteria are subjected to high temperature elimination of plasmids;
(5) And (3) taking recombinant bacteria with eliminated plasmids as host bacteria, and repeating the steps (2) - (4) with pTN-Cas9 plasmids and a vector pTK-GFP-CotB or pTC-RFP-CgeA containing another target protein to obtain the recombinant bacillus capable of simultaneously expressing green and red fluorescent proteins.
8. A recombinant Bacillus capable of simultaneously expressing a plurality of different foreign proteins, wherein the three-plasmid genome editing system of claim 1 is introduced into Bacillus belicus HCK 2.
CN202211742436.9A 2022-12-30 2022-12-30 Three-plasmid genome editing system for bacillus bailii HCK2 spore surface expression and construction and application thereof Pending CN116463370A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114806986B (en) * 2022-03-29 2023-11-14 淮阴工学院 Genetically engineered bacterium for high-yield rocmycin as well as construction method and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114806986B (en) * 2022-03-29 2023-11-14 淮阴工学院 Genetically engineered bacterium for high-yield rocmycin as well as construction method and application thereof

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