CN114525296A - Escherichia coli-pseudomonas aeruginosa shuttle expression vector and construction method thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to an escherichia coli-pseudomonas aeruginosa shuttle expression vector and a construction method thereof, wherein the escherichia coli-pseudomonas aeruginosa shuttle expression vector comprises: resistance selection genes, replication protein genes and expression region fragments; the expression region segment includes: promoter, RBS sequence, multiple cloning site, transcription terminator T of bacteriophage lambda₀Sequencing; the promoter (containing RBS sequence) and the terminator T₀The front and the back of the sequence are provided with enzyme cutting sites, the replacement of different promoter or terminator elements can be quickly realized through simple enzyme cutting and connection reaction, the steps are simple, the time for replacing the expression element of the vector is greatly shortened, and the requirements on replacing the expression element of the vector are metDiverse, high throughput cloning requirements.

Description

Escherichia coli-pseudomonas aeruginosa shuttle expression vector and construction method thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to an escherichia coli-pseudomonas aeruginosa shuttle expression vector and a construction method thereof.

Background

Rhamnolipids (Rhamnolipids) are a class of glycolipid biosurfactants, and the structure of the Rhamnolipids is composed of two parts, namely a hydrophilic group and a hydrophobic group, wherein the hydrophilic group is composed of 1-2 molecules of rhamnosyl, and the hydrophobic group is composed of 1-2 beta-hydroxy fatty acid units. Compared with the traditional chemical surfactant, the rhamnolipid not only has more excellent surface activity and stability, but also has the advantages of no toxicity, no pollution, biodegradability, good biocompatibility, good antibacterial activity, promotion of wound healing and the like, so that the rhamnolipid has unique application prospects in various fields of biological medicine, food, cosmetics, agriculture, petroleum exploitation, environmental pollution restoration and the like. With the development of society and the enhancement of environmental protection consciousness of people, the biosurfactant has wider prospect and is expected to become a substitute of a chemical synthesis surfactant.

Pseudomonas aeruginosa (Pseudomonas aeruginosa) contains an integral metabolic pathway for synthesizing rhamnolipid in vivo, and is the host which is most used for synthesizing rhamnolipid at present. However, the synthesis of rhamnolipid has the problems of low fermentation yield and high separation cost, and the industrial application of rhamnolipid is limited. Most researches adopt two ways of genetic engineering modification and fermentation process optimization (such as culture conditions, culture medium components, process control and the like) to improve the yield of rhamnolipid. Although the fermentation process optimization can obviously improve the yield of rhamnolipid, important production indexes such as conversion efficiency, fermentation substrate selection, product types and the like are still limited by the background of strain inheritance. Therefore, there is an increasing interest in developing more efficient rhamnolipid synthesis strains using genetic engineering.

In a certain range, the yield of a cell factory is directly related to the expression quantity of key synthetase, and the improvement of the expression quantity of the synthetase through the optimization of a gene expression level is an effective strategy for improving the yield of rhamnolipid. The overexpression of the rhamnolipid synthesis gene needs the participation of a vector, but the shuttle expression vector widely applied to escherichia coli-pseudomonas aeruginosa at present only comprises pAK 1900. Most studies have used a method of cloning the entire rhamnosyltransferase operon gene (rhlAB) and ligating it to a shuttle cloning vectorThe gene is overexpressed. However, the method has the problem that the natural promoter is regulated by global transcription factors such as rhlR and rhlI, so that the yield of rhamnolipid is not improved ideally. In addition, Zhao et al obtained pBBRPopraAB expression vector by connecting the constitutive promoter of oprL gene with rhlAB gene by overlap extension PCR technique; xie ET al use Red/ET DNA recombination technique to combine 3 constitutive promoters (P) with different strengths_apra、P_tacAnd P_rhaB) Replacing the original promoter of the rhlAB gene to obtain a corresponding expression vector. The construction method of the two expression vectors has complicated steps, and multiple PCR is required for replacing the promoter, so that the risk of introducing mutation in the PCR process is increased, the cloning cost is also increased, the success rate of cloning is reduced, and the requirements of diversified and high-flux cloning cannot be met. The construction of a shuttle expression vector suitable for pseudomonas aeruginosa is crucial to quickly realizing the overexpression of a target gene and improving the yield of rhamnolipid.

Therefore, a new shuttle expression vector of Escherichia coli-Pseudomonas aeruginosa and a construction method thereof need to be designed based on the technical problems.

Disclosure of Invention

The invention aims to provide an escherichia coli-pseudomonas aeruginosa shuttle expression vector and a construction method thereof.

In order to solve the technical problem, the invention provides an escherichia coli-pseudomonas aeruginosa shuttle expression vector, which comprises:

resistance selection genes, replication protein genes and expression region fragments;

the expression region segments include: promoter, multiple cloning site, transcription terminator T of bacteriophage lambda₀A sequence;

the promoter and the terminator T₀With cleavage sites before and after the sequence, and

the promoter includes the RBS sequence.

Furthermore, Hind III and BamH I cleavage sites are provided before and after the promoter.

Further, the terminator T₀Xho I and EcoR I cleavage sites are provided before and after the sequence.

Further, the RBS sequence and terminator T₀The sequences have multiple cloning site regions in between.

Further, the promoter is P_Syn8The nucleotide sequence is shown in SEQ ID NO. 1.

Further, the promoter is P_rpsJThe nucleotide sequence is shown in SEQ ID NO. 2.

Furthermore, the nucleotide sequence of the expression region fragment is shown as SEQ ID NO. 3.

Further, mScarlet gene is inserted into the multiple cloning site of the expression region fragment to form an expression vector containing the fluorescent protein gene, and the nucleotide sequence of the expression vector is shown as SEQ ID NO. 4.

Furthermore, any target gene is inserted into the multiple cloning sites of the expression region fragment to form an expression vector for expressing the corresponding target gene.

In another aspect, the invention further provides a construction method of the escherichia coli-pseudomonas aeruginosa shuttle expression vector, which comprises the following steps:

taking a cloning vector pUCP18 as a skeleton vector, and carrying out enzyme digestion to remove original multiple cloning sites;

obtaining an expression region fragment P-MCS-T through gene synthesis;

inserting the expression region fragment P-MCS-T between multiple cloning sites of a skeleton vector pUCP18 modified by enzyme digestion;

the target gene or the fluorescent indicator protein gene is inserted between the multiple cloning sites of the expression region segments.

The beneficial effect of the invention is that the expression region segments of the invention comprise: promoter (containing RBS sequence), multiple cloning site, transcription terminator T of bacteriophage lambda₀A sequence; the promoter (containing RBS sequence) and terminator T₀With restriction sites before and after the sequence, using constitutive promoter P_Syn8Or P_rpsJThe efficient and continuous transcription of the target gene in a host is ensured, and the use of an expensive inducer is avoided; the RBS sequence containing strong translation initiation sites obtained by thermodynamic calculation is introduced to ensure the high-efficiency expression of the target genes in the host. Promoter elements andthe front and the back of the terminator element are provided with enzyme cutting sites, so that the replacement of different promoters or terminator elements can be quickly realized through simple enzyme cutting and ligation reaction, the steps are simple, the time for replacing the expression element of the vector is greatly shortened, the diversified and high-flux cloning requirements are met, and a new scheme is provided for the quick construction of a promoter library; and the method for constructing the shuttle expression vector provided by the invention is also applicable to other bacteria, such as streptococcus zooepidemicus, which can be used for producing hyaluronic acid.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and drawings.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a map of the synthetic P-MCS-T expression region designed according to the present invention;

FIG. 2 is a map of a cloning vector pUCP 18;

FIG. 3 is a colony PCR map of the invention for constructing the pUCP18E expression vector;

FIG. 4 is a map of a shuttle expression vector pUCP18E constructed according to the present invention;

FIG. 5 is a colony PCR map of the invention for the construction of the pUCP18E-mSCarlet expression vector;

FIG. 6 is a map of a shuttle expression vector pUCP 18E-mSacarlet constructed according to the present invention;

FIG. 7 is the present inventionConstruction of pUCP18E-P_rpsJ-a colony PCR map of the mScarlet expression vector;

FIG. 8 shows a shuttle expression vector pUCP18E-P constructed according to the present invention_rpsJ-a map of mScarlet;

FIG. 9 shows a diagram of the present invention containing pUCP 18E-mSacarlet and pUCP18E-P_rpsJ-map of fluorescence intensity measurements of recombinant pseudomonas aeruginosa with mScarlet vector.

Detailed Description

To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present example provides an escherichia coli-pseudomonas aeruginosa shuttle expression vector, comprising: resistance selection genes, replication protein genes and expression region fragments; the expression region segments include: promoter (containing RBS sequence), multiple cloning site, transcription terminator T of bacteriophage lambda₀A sequence; the promoter (containing RBS sequence) and terminator T₀Enzyme cutting sites are arranged before and after the sequence; constructed on the basis of the Escherichia coli-Pseudomonas aeruginosa shuttle cloning vector pUCP18 and using a constitutive promoter P_Syn8Or P_rpsJThe efficient and continuous transcription of the target gene in a host is ensured, and the use of an expensive inducer is avoided; the RBS sequence of the strong translation initiation site obtained by thermodynamic calculation is introduced to ensure the high-efficiency expression of the target gene in a host. The front and back of a promoter element and a terminator element in the escherichia coli-pseudomonas aeruginosa shuttle expression vector are provided with enzyme cutting sites, so that the replacement of different promoter or terminator elements can be quickly realized through simple enzyme cutting and ligation reaction, the steps are simple, the time for replacing the vector expression element is greatly shortened, the diversified and high-flux cloning requirements are met, and meanwhile, a new scheme is provided for the quick construction of a promoter library; and the example provides a build shuttleThe method of expressing the vector is equally applicable to other bacteria, such as Streptococcus zooepidemicus, which can be used to produce hyaluronic acid.

In this example, the promoter (containing the RBS sequence) is flanked by HindIII and BamH I cleavage sites.

In the present embodiment, the terminator T₀Xho I and EcoR I cleavage sites are provided before and after the sequence.

In this example, the promoter and terminator T₀The sequences have multiple cloning site regions in between.

In this example, the promoter is P_Syn8The nucleotide sequence is shown in SEQ ID NO. 1.

In this example, the nucleotide sequence of the expression region fragment is shown in SEQ ID NO. 3.

In this example, the mScardlet gene is inserted into the multiple cloning site of the expression region fragment to form an expression vector containing the fluorescent protein gene, and the nucleotide sequence of the expression vector is shown as SEQ ID NO. 4.

In this example, the expression vector is the promoter P from which the expression vector pUCP 18E-mRecplet will be constructed_Syn8Replacement with the endogenous promoter P of Pseudomonas aeruginosa_rpsJThe nucleotide sequence is shown in SEQ ID NO. 2.

In this example, any desired gene is inserted into the multiple cloning site of the expression region fragment to construct an expression vector for expressing the desired gene.

In this example, the E.coli-P.aeruginosa shuttle expression vector had the nucleotide sequence shown in SEQ ID NO. 5.

In this example, the E.coli-P.aeruginosa shuttle expression vector contains a red fluorescent protein gene, which is used to indicate whether the target gene carried by the shuttle expression vector of this example is expressed in the host cell; the Escherichia coli-Pseudomonas aeruginosa shuttle expression vector takes a red fluorescent protein mSacrlet gene as a reporter gene, realizes the visualization of target gene expression, and solves the problems that the target gene expression level in Pseudomonas aeruginosa is low and the detection is difficult to pass through SDS-PAGE.

Specific examples are shown below, construction of the escherichia coli-pseudomonas aeruginosa shuttle expression vector pUCP 18E;

design and Synthesis of an artificially synthesized promoter P comprising Pseudomonas aeruginosa_Syn8(containing RBS sequence), multiple cloning site region and bacteriophage lambda transcription terminator T₀The gene expression region fragment P-MCS-T of the sequence. The gene expression region fragment is synthesized by Shanghai Haixin Biotechnology limited, has the length of 289bp, the nucleotide sequence shown as SEQ ID NO.3 and the sequence map shown as figure 1.

The above gene expression region fragment P-MCS-T was PCR amplified, and the PCR amplification reaction system (50. mu.L) consisted of 1. mu.L of template, 2.5. mu.L (10. mu.M) of upstream primer, 2.5. mu.L (10. mu.M) of downstream primer, 0.5. mu.L of 2X Phanta Master Mix high fidelity DNA polymerase, and 32.5. mu.L of ultrapure water. The primer sequences are as follows:

upstream primer pF 1: taaaacgacggccagtgccaagcttAGCTCTTGACAAGGTCGGAAAA

Downstream primer pR 1: gctatgaccatgattacgaattcATTCTCACCAATAAAAAACGCCCGG

PCR procedure: (1)94 ℃ for 10 min; (2)30cycles at 95 ℃ for 30 s; 30s at 55 ℃; 72 ℃ for 1 min; (3)72 ℃ for 10 min; (4)16 ℃ and +∞.

And (3) purifying the expression region fragment P-MCS-T obtained by amplification by using a DNA product gel recovery and purification kit of Shanghai Czeri bioengineering GmbH, waiting for subsequent connection, wherein the purification steps are detailed in the specification.

The cloning vector pUCP18 was double-digested with restriction enzymes Hind III and EcoR I, and was purchased from Wuhan vast Ling Biotechnology Co., Ltd, and the map is shown in FIG. 2. The linearized pUCP18 vector fragment was recovered by electrophoresis.

The obtained linearized vector fragment and the gene expression region fragment P-MCS-T are connected by adopting a one-step cloning method connection kit of Nanjing Nuojingzhan biotechnology GmbH, escherichia coli E.coli BL21(DE3) is transformed, a single clone is picked on a carbenicillin resistance plate, a shuttle expression vector is identified by PCR and sequencing, and the identification result is shown in figure 3, which indicates that the escherichia coli-pseudomonas aeruginosa shuttle expression vector is successfully constructed and is named as pUCP 18E. The shuttle expression vector is4776bp long and contains artificially synthesized promoter P of pseudomonas aeruginosa_Syn8RBS sequence, multiple cloning site region (MCS), and transcription terminator T of bacteriophage lambda₀The sequence, expression vector map is shown in figure 4, and the nucleotide sequence is shown in SEQ ID NO. 5.

Constructing an escherichia coli-pseudomonas aeruginosa shuttle expression vector pUCP 18E-mSCarlet;

the red fluorescent protein mScalet gene is amplified by PCR, the gene length is 699bp (GenBank: KY021423.1), and the sequence is synthesized by Shanghai Jiaxin Biotech limited. PCR amplification reaction (50. mu.L): mu.L of template (red fluorescent protein mScplet), 2.5. mu.L of forward primer (10. mu.M), 2.5. mu.L of reverse primer (10. mu.M), 0.5. mu.L of 2 × Phanta Master Mix high fidelity DNA polymerase, and 32.5. mu.L of ultrapure water. The primer sequences are as follows:

an upstream primer mScarlet-F: gagggggttctagagggatccATGGTCTCAAAGGGTGAAGCC

Downstream primer mScarlet-R: aacaggagtccaagactcgagTTACTTGTAAAGCTCATCCATTCCTC

PCR procedure: (1)94 ℃ for 10 min; (2)30cycles at 95 ℃ for 30 s; 30s at 55 ℃; 72 ℃ for 1 min; (3)72 ℃ for 10 min; (4)16 ℃ and +∞.

And then, purifying the obtained red fluorescent protein mSacrlet gene fragment by adopting a DNA product gel recovery and purification kit of Shanghai Czeri bioengineering GmbH, and waiting for subsequent connection, wherein the purification steps are detailed in the specification.

The shuttle expression vector pUCP18E was double-digested with restriction enzymes BamH I and Xho I, and the linearized pUCP18E vector fragment was recovered by electrophoresis.

The obtained linearized vector fragment and the red fluorescent protein mSacarlet gene fragment are connected by adopting a one-step cloning method connection kit of Nanjing Nuojingzhan biotechnology GmbH, escherichia coli E.coli BL21(DE3) is transformed, a single clone is picked on a carbenicillin resistant plate, the result of PCR and sequencing identification of the recombinant vector is shown in figure 5, the construction of the vector is proved to be correct, and the vector is named as pUCP 18E-mSacarlet. The shuttle expression vector constructed successfully has the total length of 5373bp, the expression vector map is shown in figure 6, and the nucleotide sequence is shown in SEQ ID No. 4.

Escherichia coli-pseudomonas aeruginosa shuttle expression vector pUCP18E-P_rpsJ-construction of mScarlet;

the expression vector is a promoter P of the constructed expression vector pUCP18E-mSCarlet_Syn8Replacement with the endogenous promoter P of Pseudomonas aeruginosa_rpsJThe promoter sequence is shown in SEQ ID NO. 2.

Amplifying promoter P by using pseudomonas aeruginosa PAO1 genome as template_rpsJThe length of the promoter fragment is 501 bp. PCR amplification reaction (50. mu.L): mu.L of template, 2.5. mu.L (10. mu.M) of forward primer, 2.5. mu.L (10. mu.M) of reverse primer, 0.5. mu.L of 2 XPPhanta Master Mix high fidelity DNA polymerase, and 32.5. mu.L of ultrapure water. Extracting to obtain pseudomonas aeruginosa PAO1 genome DNA by using a bacterial genome extraction kit. The primer sequences are as follows:

upstream primer P_rpsJ-F：aacgacggccagtgccaagcttagcGCACCAAGCGTGAAGACGTA

Downstream primer P_rpsJ-R：tgcaggcgcgccgagctcggatccTTTGACCTCAGACTCCAATTTACC

PCR procedure: (1)94 ℃ for 10 min; (2)30cycles at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃ C, 1

min；(3)72℃,10min；(4)16℃,+∞。

And then, purifying the obtained promoter fragment by adopting a DNA product glue recovery and purification kit of Shanghai Czeri bioengineering GmbH, waiting for subsequent connection, wherein the purification steps are detailed in the specification.

The shuttle expression vector pUCP 18E-mprecalet was double digested with restriction enzymes Blp I and BamH I, and the linearized pUCP 18E-mprecalet vector fragment was recovered by electrophoresis.

The linearized vector fragment and the promoter P_rpsJAdopting a one-step cloning method of Nanjing Nuozan biotech GmbH to connect the components, transforming Escherichia coli E.coli BL21(DE3), picking single clone on carbenicillin resistant plate, carrying out PCR and sequencing identification on recombinant expression vector, and obtaining the result shown in figure 7, wherein the result proves that the promoter sequence is successfully replaced, the expression vector is correctly constructed and is named as pUCP18E-P_rpsJ-mScarlet. The expression vector has the full length of 5817bp, the map is shown in figure 8, and the nucleotide sequence is shown in SEQ ID No. 6.

pUCP 18E-mCardet and pUCP18E-P_rpsJ-mScarlet expression verification;

mu.L of the successfully constructed shuttle expression vectors pUCP 18E-mSacarlet and pUCP18E-P were taken_rpsJ-mscale, sequentially added to pseudomonas aeruginosa ATCC 27853 competent cells, pipetted well and incubated on ice for 30 min. A BIO-RAD Micro Pulser electric converter is used, a 0.1cm electric shock cup is selected for conversion, the electric shock voltage is 1.6kV, the time is 2.0ms, the electric shock cup is placed on ice after the electric shock is finished, 800 mu L of LB culture medium is rapidly added, after the blowing, sucking and re-suspending, the sucked bacterial liquid is placed in a centrifuge tube, the bacterial liquid is subjected to shake culture in a shaking table at 37 ℃ for 2 hours, then the bacterial liquid is coated on an LB flat plate containing carbenicillin resistance, a single bacterial colony is selected to be cultured in the LB culture medium containing the carbenicillin resistance at 37 ℃, an appropriate amount of bacterial liquid is taken next day, 30% of glycerol is added, and the bacterial liquid is placed in a refrigerator at-80 ℃ for storage.

And respectively inoculating 5 mu L of the pseudomonas aeruginosa mSacrle recombinant bacteria into 30mL of LB culture medium, culturing at 37 ℃ overnight at 220rpm, sucking 1mL of bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 12000rpm for 1min, removing the supernatant, and observing that the bacteria are red. Resuspending the bacteria with 200. mu.L of distilled water, shaking and mixing, and respectively measuring OD on a Microplate reader₆₀₀And the fluorescence intensity of the red fluorescent protein mScalet, the maximum excitation wavelength is 569nm, the maximum emission wavelength is 594nm, data are collected, and the promoter P is drawn_Syn8And P_rpsJThe result of the fluorescence intensity map of the transcribed mSacarlet is shown in FIG. 9, and it can be seen from the figure that the pUCP18E vector can correctly express the red fluorescent protein mSacarlet gene, and the promoter is replaced by the endogenous promoter P of the pseudomonas aeruginosa_rpsJThe mSacrlet fluorescent protein gene can also be successfully expressed later. The feasibility of the vector construction method and the promoter replacement method is proved.

The embodiment also provides a construction method of the shuttle expression vector of escherichia coli-pseudomonas aeruginosa, which comprises the following steps: taking a cloning vector pUCP18 as a skeleton vector, and carrying out enzyme digestion to remove original multiple cloning sites; obtaining an expression region fragment P-MCS-T through gene synthesis; inserting the expression region fragment P-MCS-T between multiple cloning sites of a skeleton vector pUCP18 modified by enzyme digestion; the target gene or the fluorescent indicator protein gene is inserted between the multiple cloning sites of the expression region segments.

In conclusion, the invention uses Escherichia coli-Pseudomonas aeruginosa shuttle cloning vector pUCP18 as the basis to replace pUCP18 inherent multiple cloning site region and introduce promoter P_Syn8Or P_rpsJ(containing RBS sequence), MCS sequence and bacteriophage lambda transcription terminator T₀The sequence successfully constructs shuttle expression vectors pUCP18E, pUCP 18E-mSacrlet and pUCP18E-P of Escherichia coli-pseudomonas aeruginosa_rpsJThe escherichia coli-pseudomonas aeruginosa shuttle expression vector has Hind III and BamH I enzyme cutting sites before and after a promoter (containing an RBS sequence) and Xho I and EcoR I enzyme cutting sites before and after a terminator sequence, can simply, conveniently and quickly replace any promoter and terminator sequence, and meets the requirements of diversified and high-flux cloning; can be widely applied to the expression of all exogenous genes taking pseudomonas aeruginosa as host bacteria and the production of gene engineering products thereof, provides a new choice for the application of related fields, and provides a new expression vector tool for the related application of the fields. The construction method of the shuttle expression vector can also be applied to the design and construction of other host shuttle expression vectors, and has good application prospect.

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims (10)

1. An escherichia coli-pseudomonas aeruginosa shuttle expression vector comprising:

resistance selection genes, replication protein genes and expression region fragments;

the expression region segments include: promoter, multiple cloning site, transcription terminator T of bacteriophage lambda₀A sequence;

the promoter and the terminator T₀With cleavage sites before and after the sequence, and

the promoter includes the RBS sequence.

2. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of claim 1,

the promoter is provided withHind III andBamHI enzyme cleavage siteAnd (4) point.

3. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of claim 1,

the terminator T₀With preceding and following sequenceXhoI andEcor I enzyme cutting site.

4. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of claim 1,

the promoter and the terminator T₀The sequences have multiple cloning site regions in between.

5. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of claim 1,

the promoter is P_Syn8The nucleotide sequence is shown in SEQ ID NO. 1.

6. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of claim 1,

the promoter is P _rpsJ The nucleotide sequence is shown in SEQ ID NO. 2.

7. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of claim 1,

the nucleotide sequence of the expression region fragment is shown as SEQ ID NO. 3.

8. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of any one of claims 4 to 6,

and inserting the mScarlet gene into the multiple cloning site of the expression region fragment to form an expression vector containing the fluorescent protein gene, wherein the nucleotide sequence of the expression vector is shown as SEQ ID NO. 4.

9. The Escherichia coli-Pseudomonas aeruginosa shuttle expression vector of any one of claims 4 to 6,

inserting any target gene into the multiple cloning site of the expression region segment to form an expression vector for expressing the corresponding target gene.

10. A method for constructing the escherichia coli-pseudomonas aeruginosa shuttle expression vector according to claim 1, comprising:

taking a cloning vector pUCP18 as a skeleton vector, and carrying out enzyme digestion to remove original multiple cloning sites;

obtaining an expression region fragment P-MCS-T through gene synthesis;

inserting the expression region fragment P-MCS-T between multiple cloning sites of a skeleton vector pUCP18 modified by enzyme digestion;

the target gene or the fluorescent indicator protein gene is inserted between the multiple cloning sites of the expression region segments.

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