CN116875595A - BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum and preparation method thereof - Google Patents
BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum and preparation method thereof Download PDFInfo
- Publication number
- CN116875595A CN116875595A CN202310617466.5A CN202310617466A CN116875595A CN 116875595 A CN116875595 A CN 116875595A CN 202310617466 A CN202310617466 A CN 202310617466A CN 116875595 A CN116875595 A CN 116875595A
- Authority
- CN
- China
- Prior art keywords
- plasmid
- bdp
- bdps
- corynebacterium glutamicum
- switch
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102100025799 Protein phosphatase 1K, mitochondrial Human genes 0.000 title claims abstract description 60
- 241000186226 Corynebacterium glutamicum Species 0.000 title claims abstract description 43
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 238000012512 characterization method Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000013612 plasmid Substances 0.000 claims abstract description 92
- 241000282326 Felis catus Species 0.000 claims abstract description 11
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 33
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- 239000012634 fragment Substances 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 235000019445 benzyl alcohol Nutrition 0.000 claims description 11
- 230000006801 homologous recombination Effects 0.000 claims description 11
- 238000002744 homologous recombination Methods 0.000 claims description 11
- 238000011144 upstream manufacturing Methods 0.000 claims description 11
- 238000010276 construction Methods 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 230000001965 increasing effect Effects 0.000 claims description 7
- 230000009466 transformation Effects 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 6
- 230000037431 insertion Effects 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 230000002457 bidirectional effect Effects 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract description 8
- 238000009412 basement excavation Methods 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000003776 cleavage reaction Methods 0.000 description 8
- 230000007017 scission Effects 0.000 description 8
- 230000006698 induction Effects 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010037936 CCCGGG-specific type II deoxyribonucleases Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940060587 alpha e Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000005558 fluorometry Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/265—Micrococcus
- C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a novel BDPs activity characterization biosensor BDP-SWITCH in corynebacterium glutamicum and a preparation method thereof, wherein the biosensor comprises a BDP cat And BDP icl A plasmid of the promoter. The application provides a fluorescence biosensor capable of representing BDPs intensity for high-flux screening, which provides an effective tool for high-flux excavation of natural BDPs by corynebacterium glutamicum and provides a new idea for BDPs screening in prokaryotes.
Description
Technical Field
The application belongs to the technical field of bioengineering, and particularly relates to a novel BDP-SWITCH for representing BDPs activity in corynebacterium glutamicum and a preparation method thereof.
Background
BDPs (bidirectional promoters, BDPs) are a DNA sequence located between two adjacent genes with opposite transcription directions, i.e., a DNA sequence located between two "head-to-head" gene pairs. The main methods for obtaining the novel promoter based on the genome level at home and abroad at present comprise a promoter probe carrier screening method, a conventional PCR method, a sequence specific primer PCR method, a genome library screening method and the like. However, due to the structural specificity of BDPs, BDPs screening and promoter strength identification have certain limitations. The most commonly used identification modes are single or small in BDPs intensity verification, and have low efficiency in application of genome-level high-throughput BDPs screening, and obvious limitations.
Corynebacterium glutamicum has been increasingly used as a cell factory and a heterologous protein expression host, but the problem of few selectable expression elements in constructing a gene loop has not been solved. The conventional work of excavating, modifying and rationally designing a unidirectional promoter of corynebacterium glutamicum has a rich research background, but the excavating method of BDPs and the excavating method of high-throughput BDPs at the genome level have not been reported in related literature. Due to the specific structure of BDPs, the excavation methods commonly used for MDPs are obviously not suitable for the excavation of BDPs.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present application has been made in view of the above and/or problems occurring in the prior art.
Therefore, the aim of the application is to overcome the defects in the prior art and provide a novel biosensor BDP-SWITCH for representing BDPs activity in corynebacterium glutamicum.
In order to solve the technical problems, the application provides the following technical scheme: BDPs activity in Corynebacterium glutamicum characterizes a novel biosensor BDP-SWITCH, which comprises, the biosensor comprises, the p19BDP plasmid.
As a preferred scheme for characterizing BDPs activity in Corynebacterium glutamicum according to the application, the novel biosensor BDP-SWITCH, wherein: BDP contained in plasmid cat And BDP icl The genes upstream and downstream of the promoter are arranged in a "head-to-head" configuration.
Another object of the application is to provide a method for preparing BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum.
In order to solve the technical problems, the application provides the following technical scheme: a preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum comprises the following steps,
preparing a plasmid: the gSTEP1 was introduced into the plasmid PEC to obtain plasmid pEC-gSTEP1, and plasmids pXMJ19-STEPtag, plasmid p19BDP, plasmid p19-catMF-gSTEP1, plasmid p19-catMR-STEPtag, plasmid p19BDP-cat and p19BDP-icl were prepared in the same manner as the manner of gene introduction
Transformation of Corynebacterium glutamicum: introducing plasmids p19BDP-cat and p19BDP-icl into competent cells of corynebacterium glutamicum, and culturing in a culture medium containing corresponding resistance after transformation to obtain a transformant;
fermentation culture: inoculating the transformant, and culturing to obtain a large number of strains;
constructing a sensor model plasmid: the p19-0 plasmid is taken as a plasmid skeleton, restriction endonuclease SmaI is used for digestion, the gSTEP1 and STEPtag gene sequences are inserted into the plasmid, an M13 sequence is inserted downstream of the riboJ for homologous recombination, and SmaI is selected as an insertion site.
As a preferred scheme for the preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum, the application comprises the following steps: in constructing the sensor model plasmid, the gsep 1 and the STEPtag gene sequences are inserted into the plasmid in a recombinant mode according to a head-to-head structure.
As a preferred scheme for the preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum, the application comprises the following steps: in the preparation of the plasmids, the target genes of the corresponding introduced plasmids are all amplified by adopting forward primers, and the plasmids into which the target genes are introduced are obtained by homologous recombination with the corresponding plasmids after the amplification.
As a preferred scheme for the preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum, the application comprises the following steps: in the transformation of corynebacterium glutamicum, the medium is LBHIS solid medium.
As a preferred scheme for the preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum, the application comprises the following steps: plasmid p19BDP-cat is obtained by introducing Pcat sequence fragment and Picl sequence fragment into one plasmid.
As a preferred scheme for the preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum, the application comprises the following steps: all plasmids produced showed an increase in fluorescence with increasing concentrations of benzyl alcohol and ethanol alone, and a significantly higher value for fluorescence intensity than without benzyl alcohol and ethanol.
As a preferred scheme for the preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum, the application comprises the following steps: the prepared plasmid has no obvious enhancement of fluorescence when BDP-SWITCH sensor carries out unidirectional expression under the environment of benzyl alcohol and ethanol, and has obvious enhancement of fluorescence intensity when facing bidirectional expression.
The application has the beneficial effects that:
the research is based on the principle that green fluorescence expression can be activated by the interaction of green fluorescence transient characterization protein sensor protein 1 (green fluorescent sensor for transiently expressed proteins, gSTEP 1) protein and transient characterization protein sensor protein labels (sensor for transiently expressed proteins tag, STEPtag) to form a gSTEP1/STEPtag complex, a fluorescence biosensor capable of being used for high-flux screening and representing BDPs intensity is rationally designed, an effective tool is provided for high-flux mining of natural BDPs by corynebacterium glutamicum, and a new thought is provided for screening BDPs in prokaryotes.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a schematic representation of plasmid p19 and the corresponding subsequent plasmids;
in the figure, a is a schematic diagram of a p19 plasmid and an XhoI cleavage site inserted into the plasmid; b is a schematic diagram after insertion of SmaI cleavage site; c is the plasmid structure obtained after completion of example 4;
FIG. 2 shows the structure of plasmids p19-catMF-gSTEP1 and p 19-catMR-STEPtag;
FIG. 3 shows an ethanol-inducible BDP prepared according to the application icl Analyzing the fluorescence value of the gradient induction concentration;
FIG. 4 shows a benzyl alcohol-induced BDP prepared by the present application cat Gradient induced concentration fluorescence values;
wherein, WT-wild type C.g1.15647; MF-C.g1.15647 containing the p19-catMF-STEP1 plasmid; MR-containing p19-catMR-STEP1 plasmid C.g1.15647; B-C.g 1.15647 containing the p19BDP-cat plasmid; NS: no significant differences were found.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The culture medium used in the examples of the present application is as follows:
LB medium (g/L): yeast extract 5.0, peptone 10.0, sodium chloride 10.0; corynebacterium glutamicum culture Medium LBB Medium (g/L): yeast extract 5.0, peptone 10.0, sodium chloride 10.0, brain-heart extract 10.0;
corynebacterium glutamicum recovery Medium LBHIS Medium (g/L): yeast extract 2.5, peptone 5.0, sodium chloride 10.0;
preparation of C.glutamicum competent Medium NCM Medium (g/L): yeast extract 1.0, peptone 5.0, sodium chloride 11.6, dipotassium hydrogen phosphate 17.4, glucose 5.0, trisodium citrate 0.3, magnesium sulfate 0.05, sorbitol 91.0, and adjusting the pH to 7.2.
2% agar was added when preparing the solid medium. The final concentration of chloramphenicol was 30mg/L in E.coli, 10mg/L in C.glutamicum medium, and 50mg/L in E.coli and C.glutamicum medium.
Example 1
This example was used to construct plasmids:
the gene sequences of gsep 1 and STEPtag are synthesized by Jin Wei intelligent company by codon optimization according to the preference of the corynebacterium glutamicum codon; all plasmids are obtained by PCR amplification to obtain fragments and the recombination of DH5 alpha E.coli competent cells after the SmaI restriction endonuclease cuts the vector, positive clones are screened on LB plates containing corresponding resistance, and the plasmids are extracted and sent to Suzhou Jin Weizhi company for sequencing.
1. Construction of plasmid pEC-gSTEP 1: using the synthesized sequence gSTEP1 as a template, and carrying out PCR amplification by using forward and reverse primers RBS-gSTEP1-F and gSTEP1-R (respectively shown as SEQ ID NO.3 and SEQ ID NO. 4) to obtain a gSTEP1 sequence fragment (shown as SEQ ID NO. 1), wherein the PCR parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃ for 30s;20 cycles; the plasmid pEC-gSTEP1 was prepared by amplifying the p19-H36-riboJ-sfGFP plasmid as a template with the primer H36-riboJ-RBS-F, riboJ-RBS-R (shown as SEQ ID NO.5 and SEQ ID NO.6, respectively) to obtain the H36-riboJ-RBS sequence, and ligating the gSTEP1 sequence, the H36-riboJ-RBS sequence and the linearized plasmid backbone pEC-xk99e by homologous recombination.
2. Construction of plasmid pXMJ 19-STEPtag: using synthetic sequence gstePtag as template, using forward and reverse primer RBS-STEPtag-F, STEPtag-his-R (shown as SEQ ID NO.7 and SEQ ID NO.8 respectively), PCR amplifying to obtain STEPtag sequence fragment (shown as SEQ ID NO. 2), wherein PCR parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s;20 cycles; the pXMJ19-STEPtag is obtained by connecting STEPtag sequences and linearized plasmid backbone pXMJ19 by homologous recombination.
3. Construction of plasmid p19 BDP: the ribozyme gene sequence fragment ElvJ is obtained by PCR of an upstream primer ElvJ-syn-F, elvJ-syn-R (shown as SEQ ID NO.9 and SEQ ID NO.10 respectively), and the PCR parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃ for 30s;20 cycles. The plasmid pXMJ19-STEPtag is used as a template, and a primer MCS-his-STEPtag-F, elvJ-RBS-STEPtag-R (shown as SEQ ID NO.11 and SEQ ID NO.12 respectively) is utilized for amplification to obtain STEPtag sequence fragments; using the ElvJ sequence and STEPtag sequence as templates, and amplifying by using a primer M13rev-riboJ-F, elvJ-RBS-STEPtag-R (shown as SEQ ID NO.13 and SEQ ID NO.12 respectively) to obtain an ElvJ-STEPtag fragment; using plasmid pEC-gSTEP1 as template, using primers H36-riboJ-RBS-F and ElvJ-M13fwd-R (shown as SEQ ID NO.5 and SEQ ID NO.14 respectively), PCR amplifying to obtain riboJ-gSTEP1 sequence fragment, wherein the PCR parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s;20 cycles. Plasmid p19BDP was prepared by joining the ElvJ-STEPtag sequence, the riboJ-gSTEP1 sequence and the linearized plasmid backbone p19-0 by homologous recombination.
4. Construction of plasmid p19-catMF-gSTEP 1: cleavage of plasmid p19-0; PCR amplification is carried out by taking plasmid pEC-gSTEP1 as a template and utilizing forward and reverse primers RBS-gSTEP1-F and gSTEP1-R (shown as SEQ ID NO.3 and SEQ ID NO.4 respectively) to obtain a riboJ-gSTEP1 sequence fragment; the forward (defined herein) promoter P was obtained by PCR amplification using C.g1.15647 genome as template and the upstream and downstream primers cat-MF-F, cat-MF-R (shown as SEQ ID NO.15 and SEQ ID NO.16, respectively) catMF Sequence fragments, PCR parameters of 95 ℃ denaturation 30s,56 ℃ annealing 30s,72 ℃ extension 30s;20 cycles; ligation of P by homologous recombination catMF The sequence, the riboJ-gsteP1 sequence and the linearized vector p19BDP were obtained to give the plasmid p19-catMF-gsteP1.
5. Construction of plasmid p 19-catMR-STEPtag: cleavage of plasmid p19-0; the plasmid pXMJ19-STEPtag is used as a template, forward and reverse primers RBS-STEPtag-F, STEPtag-his-R (shown as SEQ ID NO.7 and SEQ ID NO.8 respectively) are utilized, and the sequence fragment of the ELvJ-STEPtag is obtained through PCR amplification, wherein the PCR parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃ for 30s; the genome C.g1.15647 is used as a template, and a forward and reverse primer cat-MR-F, cat-MR-R (shown as SEQ ID NO.17 and SEQ ID NO.18 respectively) is utilized for PCR amplification to obtain a reverse promoter P catMR Sequence fragments, joined P by homologous recombination catMR The sequence, the ElvJ-STEPtag sequence and the linearization vector p19BDP are obtained to prepare the plasmid p19-catMR-STEPtag.
6. Construction of plasmid p19BDP-cat and p19 BDP-icl: cleavage of plasmid p19BDP; the genome is used as a template, and the primers are respectively used for amplification to obtain P cat Sequence fragment and P icl Sequence fragments; and respectively recombining the promoter fragment and the vector after enzyme digestion to obtain plasmids p19BDP-cat and p19BDP-icl.
Example 2
This example was used to carry out the transformation of Corynebacterium glutamicum:
1 mu g p BDP-cat plasmid was added to 100. Mu.L C.g1.15647 competent cells, ice-bath for 5-10min,1.8kV,5ms shocked twice, and then to pre-heated 1mL LBHIS medium, water bath at 46℃for 6min. After culturing for 2 hours at 30 ℃ by a shaking table, the transformant can be grown by coating on a LBHIS solid culture medium plate with corresponding resistance and then standing and culturing for 36-48 hours at 30 ℃.
Example 3
This example was used to perform fermentation of Corynebacterium glutamicum and preparation of the Corynebacterium glutamicum for fluorometry:
the single colony with good growth prepared in example 2 was picked up and inoculated into LBB liquid medium for cultivation at 30℃and 220r/min for 12 hours. The seed culture solution is subjected to initial OD 600 Inoculum size of =0.2 was transferred to LBB liquid medium (inducible promoter contains appropriate concentration of corresponding inducer) and cultured at 30 ℃ for 24h at 220 r/min.
The cultured bacterial liquid needs to be diluted by a culture medium, and the final concentration OD of the diluted liquid 600 =0.2-0.6, and OD of bacterial liquid was measured by Bio Tek multifunctional enzyme-labeled instrument 600 And fluorescence intensity (excitation wavelength: 488 nm, emission wavelength: 520 nm). At OD of each bacterial liquid sample 600 The relative Fluorescence value is calculated as Fluorescence/OD 600 For specific determination see example 5.
Example 4
This example was used to construct BDP-SWITCH sensor model plasmid p19BDP
The p19-0 plasmid was used as the plasmid backbone, digested with restriction endonuclease SmaI, the gSTEP1 and STEPtag gene sequences were recombinantly inserted into the plasmid in a "head-to-head" configuration, and XhoI cleavage sites were inserted into the fragments for BDPs fragment insertion (FIG. 1 a). Insertion of the BDPs fragments after construction was found to be unable to obtain the p19BDP-BDPn plasmid of interest. The reason is probably because RiboJ and EvlJ are homologous sequences and RNA structures are easily circularized to form clover structures, so homologous recombination is difficult to accomplish when RiboJ and EvlJ recombination homology arms are contained upstream and downstream of BDPs fragments. Therefore, a spacer sequence (ATGCCTCCACACCGCTCGTCACATCCTG) was inserted downstream of the riboJ for homologous recombination with a positive rate of 60%. The positive rate is improved to 80% by changing the insertion cleavage site to SmaI, probably because SmaI is blunt-ended, which is more favorable for the action of the recombinase. Considering plasmid verification and sequencing requirements, the spacer sequence is replaced by a common M13 sequence (GTAAAACGACGGCCAGTCCCGGGGTCATAGCTGTTTCCTG), and a SmaI cleavage site is inserted in the middle of the upstream and downstream of the spacer sequence (FIG. 1 b), so that the final recombination positive rate reaches 98%. The reason is probably that the upstream and downstream sequences of M13 are used as recombination homology arms to completely replace the riboJ and EvlJ sequences, and the influence of a circular structure and a homologous sequence on the action of the recombinase is not generated. The final plasmid structure is shown in FIG. 1 c.
Example 5
This example was used to perform the validation of BDP-SWITCH sensors by inducible endogenous BDPs
Analysis C.g1.15647 of two known inducible promoters P based on transcriptional data cat (benzyl alcohol inducible) and P icl (ethanol inducible), the genes upstream and downstream of the two promoters were found to be in a "head-to-head" configuration, thus presumably having bidirectional promoter activity, respectively designated BDP cat And BDP icl To distinguish it from the corresponding MDPs.
Using the P19-0 vector as a framework, control vectors P19-catMF-STEP1 and P19-catMR-STEPtag were constructed to characterize P cat Fluorescence values generated by unidirectional expression of the target protein in both directions (FIG. 2). Using C.g1.15647 genome as template, designing primer for amplification to obtain BDP cat BDP icl The sequences, recombinant to obtain the probe vector p19BDP-cat and p19BDP-icl. Plasmid p19BDP-cat and plasmid p19BDP-icl were transformed and induced by addition of gradient concentration inducer. BDP under 0-8-mM benzyl alcohol induction condition cat Unit OD after 24h fermentation 600 Fluorescence intensity increased with increasing inducer concentration, unit OD at 8mM 600 The fluorescence intensity reached a maximum of 329689.3 (fig. 4), 15.9 times before induction. BDP under 0-1% (v/v) ethanol induction conditions icl Unit OD after 24h fermentation 600 Fluorescence intensity increased with increasing inducer concentration, 1% (v/v) unit OD 600 The fluorescence intensity reached the highest value 490458.2, 6.2 times before induction (fig. 3). According toAs a result, subsequent experiments selected 8mM benzyl alcohol and 1% (v/v) ethanol for induction. The unit OD600 fluorescence values obtained by fermentation of p19-catMF-STEP1, p19-catMR-STEPtag, and p19BDP-cat at a benzyl alcohol concentration of 8mM are shown in FIG. 4, respectively. The fluorescence values of p19BDP-cat after induction and fermentation for 24h are 17 times of that of p19-catMF-STEP1 and 22 times of that of p19-catMR-STEPtag respectively, and compared with a control (C.g 15647 containing p 19-0), the fluorescence value of p19-catMF-STEP1 is only increased by 0.1 times, and the fluorescence of p19-catMR-STEPtag hardly emits. The probe carrier has no obvious enhancement of fluorescence when the promoter is expressed unidirectionally. The results indicate that BDP-SWITCH sensors can be used to mine and characterize the intensity of BDPs.
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Claims (9)
1. The BDPs activity in corynebacterium glutamicum characterizes a novel biosensor BDP-SWITCH, which is characterized in that: included are biosensors including p19BDP-cat and p19BDP-icl, i.e.biosensors containing both cat and icl genes.
2. The activity characterization of BDPs in corynebacterium glutamicum according to claim 1, characterized in that it is a novel biosensor BDP-SWITCH: BDP contained in the plasmid cat And BDP icl The genes upstream and downstream of the promoter are arranged in such a manner that the upstream is upstream to the upstream and the downstream is downstream to the downstream.
3. A preparation method of BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum is characterized by comprising the following steps: comprises the following steps of the method,
preparing a plasmid: the gSTEP1 was introduced into the plasmid PEC to obtain a plasmid pEC-gSTEP1, and the transformation of the plasmids pXMJ19-STEPtag, p19BDP, p19-catMF-gSTEP1, p19-catMR-STEPtag, p19BDP-cat and p19BDP-icl Corynebacterium glutamicum was similarly prepared by the way of gene introduction: introducing plasmids p19BDP-cat and p19BDP-icl into competent cells of corynebacterium glutamicum, and culturing in a culture medium containing corresponding resistance after transformation to obtain a transformant;
fermentation culture: inoculating the transformant, and culturing to obtain a large number of strains;
constructing a sensor model plasmid: the p19-0 plasmid is taken as a plasmid skeleton, restriction endonuclease SmaI is used for digestion, the gSTEP1 and STEPtag gene sequences are inserted into the plasmid, an M13 sequence is inserted downstream of the riboJ for homologous recombination, and SmaI is selected as an insertion site.
4. A method for the preparation of BDPs activity characterization of novel biosensor BDP-SWITCH in corynebacterium glutamicum according to claim 3, characterized in that: in the construction of the sensor model plasmid, the gsep 1 and the STEPtag gene sequences are inserted into the plasmid in a recombinant mode according to a head-to-head structure.
5. A method for the preparation of BDPs activity characterization of novel biosensor BDP-SWITCH in corynebacterium glutamicum according to claim 3, characterized in that: in the preparation of the plasmids, the target genes of the corresponding introduced plasmids are all amplified by adopting forward primers, and the plasmids into which the target genes are introduced are obtained by homologous recombination with the corresponding plasmids after amplification.
6. A method for the preparation of BDPs activity characterization of novel biosensor BDP-SWITCH in corynebacterium glutamicum according to claim 3, characterized in that: in the transformation of corynebacterium glutamicum, the culture medium is LBHIS solid culture medium.
7. A method for the preparation of BDPs activity characterization of novel biosensor BDP-SWITCH in corynebacterium glutamicum according to claim 3, characterized in that: the plasmid P19BDP-cat is obtained by introducing a Pcat sequence fragment into a plasmid, and the plasmid P19BDP-icl is obtained by introducing a Picl sequence fragment into the plasmid.
8. The method for preparing BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum according to any one of claims 3 to 7, characterized in that: all of the plasmids produced showed an increase in fluorescence with increasing concentrations of benzyl alcohol and ethanol alone, and a significantly higher value for fluorescence intensity than without benzyl alcohol and ethanol.
9. The method for preparing BDPs activity characterization novel biosensor BDP-SWITCH in Corynebacterium glutamicum according to claim 8, wherein: the prepared plasmid has no obvious enhancement of fluorescence when the BDP-SWITCH sensor carries out unidirectional expression under the environment with benzyl alcohol and/or ethanol, and has obvious enhancement of fluorescence intensity when facing bidirectional expression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310617466.5A CN116875595A (en) | 2023-05-29 | 2023-05-29 | BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310617466.5A CN116875595A (en) | 2023-05-29 | 2023-05-29 | BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116875595A true CN116875595A (en) | 2023-10-13 |
Family
ID=88253580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310617466.5A Pending CN116875595A (en) | 2023-05-29 | 2023-05-29 | BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116875595A (en) |
-
2023
- 2023-05-29 CN CN202310617466.5A patent/CN116875595A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112080521B (en) | Recombinant pseudorabies virus vector construction for expressing foreign protein and preparation method of recombinant pseudorabies virus | |
| CN109477115A (en) | For Eukaryotic expression system | |
| CN110079546B (en) | Multi-gene knock-in method for pichia pastoris expression host | |
| US20230272374A1 (en) | Compositions and methods for identifying polynucleotides of interest | |
| CN113061608B (en) | Evolution method of inducible promoter and application thereof | |
| Wu et al. | An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei | |
| CN110628738B (en) | Method for improving activity of glucose oxidase, mutant and application thereof | |
| US20140178933A1 (en) | Enhanced heterologous protein production in kluyveromyces marxianus | |
| CN106701787A (en) | Pichia pastoris for expressing foreign proteins, construction method of pichia pastoris and induced expression method of pichia pastoris | |
| US20110129874A1 (en) | Pichia Pastoris Das Promoter Variants | |
| Wu et al. | Expression of the gene coding for bacterial hemoglobin improves β-galactosidase production in a recombinant Pichia pastoris | |
| CN110205334B (en) | System and method for performing multi-round editing on fungal genome through CRISPR (clustered regularly interspaced short palindromic repeats) system | |
| CN116875595A (en) | BDPs activity characterization novel biosensor BDP-SWITCH in corynebacterium glutamicum and preparation method thereof | |
| JP2973430B2 (en) | Method for producing protein by yeast using inducible system, vector and transformant thereof | |
| CN112899296A (en) | Transposase screening report vector and preparation method and application thereof | |
| CN108060168B (en) | Improved promoter, T vector composed of improved promoter and application of improved promoter | |
| CN114107360B (en) | Method for improving cellulase expression of trichoderma reesei by interfering phosphatase gene | |
| Papagianni et al. | Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions | |
| CN108148853B (en) | Genome editing vector, genome editing system composed of same and application of genome editing system | |
| CN104513830A (en) | Gene expression vector applicable to gluconobacter oxydans and application of gene expression vector | |
| CN110628799A (en) | Construction method and application of bacterial promoter report vector | |
| CN106480031B (en) | Bacillus expresses promoter and its application | |
| CN105505933B (en) | Novel arthrobacterium promoter sequence and its application | |
| CN112831517B (en) | Lycopene gene-mediated modification cloning vector and application thereof | |
| CN112680449B (en) | Ustilago esculenta endogenous promoter pEF as well as expression vector and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |