Transposon system without resistance screening label residue and application thereof

Abstract

The invention relates to a transposon system without resistance screening label residues and application of the transposon system. The transposon system is a single-plasmid transposon system or a double-plasmid transposon system. The single plasmid transposon system comprises a first plasmid which expresses transposase and carries a reporter gene and a ptxD gene; the double plasmid transposon system comprises a second plasmid for expressing transposase and a third plasmid carrying a reporter gene and a ptxD gene. The transposon system utilizes the ptxD screening tag, gene integration without antibiotic resistance gene residue can be realized, potential safety hazards and metabolic burden caused by resistance screening tag residue can be avoided, and the transposon system has higher competitiveness in actual production application.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a transposon system without residual resistance screening tags and its application. Background Art

[0002] The transposon integration technology is one of the common methods for editing the genome of engineered bacteria. Under the action of transposase, a target gene with specific recognition sequences at both ends can be inserted into the host genome to achieve stable gene integration. This technology plays an important role in many fields such as gene function research, transgenic organism construction, and gene therapy. Usually, the transposon system screens strains with integrated target genes through resistance screening tags, and the residual antibiotic resistance genes will increase the metabolic burden of the host, bring the potential risk of antibiotic resistance gene transfer, and at the same time limit the application of engineered bacteria in the fields of medicine, agriculture, etc.

[0003] Therefore, how to make the transposon system get rid of the limitation of resistance screening tags remains to be studied. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide a transposon system without residual resistance screening tags and its application. This transposon system utilizes the ptxD screening tag and can achieve gene integration without residual antibiotic resistance genes.

[0005] To this end, on the one hand of the present invention, the present invention proposes a transposon system without residual resistance screening tags, and this transposon system is a single plasmid transposon system or a two plasmid transposon system;

[0006] The single plasmid transposon system includes a first plasmid that expresses transposase and carries a reporter gene and a ptxD gene;

[0007] The two plasmid transposon system includes a second plasmid that expresses transposase and a third plasmid that carries a reporter gene and a ptxD gene.

[0008] According to the transposon system without residual resistance screening tags of the present invention, phosphite oxidoreductase (PTXD) is encoded by the ptxD gene, and this enzyme can convert phosphite (Phi) into phosphate (Pi); using the ptxD gene as the screening tag of the transposon system, strains that exogenously express the ptxD gene can convert phosphite. By combining a medium with phosphite as the sole phosphorus source, transposon system edited strains without residual resistance screening tags can be obtained, avoiding the residual antibiotic resistance genes, so that the transposon system can be applied to intestinal probiotics and reduce the impact on the intestinal flora.

[0009] In addition, the transposon system without residual resistant screening tags proposed according to the above embodiments of the present invention may further have the following additional technical features:

[0010] Optionally, the reporter gene is the enhanced green fluorescent protein eGFP gene, and other production modules can be added after eGFP to obtain production strains more efficiently.

[0011] Optionally, the transposon system is a non-replicative Tn5 transposon system. Among them, the transposon system can also be Himar1, PiggyBac, etc.

[0012] Optionally, the pKD3-Cm-Tn5 vector with the nucleotide sequence shown in SEQ ID NO:1 is cut by NotⅠ-XhoⅠ, and ligated with the reporter gene and the ptxD gene module P with the nucleotide sequence shown in SEQ ID NO:2 after being cut by NotⅠ-XhoⅠ con -eGFP-ptxD to obtain the pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 plasmid, which is the third plasmid;

[0013] The pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 plasmid is cut by XbaⅠ and ligated with the transposase gene module P with the nucleotide sequence shown in SEQ ID NO:5 after being cut by XbaⅠ tetA -tnp* to obtain the pKD3-Cm-P tetA -tnp*-Tn5-P con -eGFP-ptxD-Tn5 plasmid, which is the first plasmid.

[0014] Optionally, the pSC101 vector with the nucleotide sequence shown in SEQ ID NO:3 is cut by EcoRⅠ-SalⅠ and ligated with the transposase gene module P with the nucleotide sequence shown in SEQ ID NO:4 after being cut by EcoRⅠ-SalⅠ tetA -tnp to obtain the pSC101-tnp plasmid, which is the first plasmid.

[0015] In the second aspect of the present invention, the present invention proposes a method for editing strains using the above transposon system without residual resistant screening tags, including:

[0016] Randomly integrating the transposon system into the genome of the target host;

[0017] Screen the target host on a plate with phosphite as the sole phosphorus source to obtain colonies that can grow normally, thereby obtaining strains with successful gene integration. At the same time, the output intensity of the reporter gene is used as an indicator of the expression level of the target gene to judge the integration effect.

[0018] According to a method for editing strains using the transposon system without residual resistance screening tags of the present invention, the ptxD gene is used as the screening tag of the transposon system. Under the screening pressure with phosphite as the sole phosphorus source, transposon strains without resistance can be rapidly obtained. Compared with traditional transposon systems, due to the limitation of resistance tags, most transposons cannot be practically applied to subsequent intestinal therapy or production practice applications. Therefore, the method for editing strains using the transposon system without residual resistance screening tags is more competitive in practical applications.

[0019] Optionally, the target host is Escherichia coli Nissle 1917, or it can also be other chassis cells.

[0020] Optionally, transform the first plasmid into Escherichia coli Nissle 1917 competent cells, screen the strains that grow normally on a plate with phosphite as the sole phosphorus source, randomly pick colonies, detect green fluorescence, and judge the gene integration effect of the single plasmid transposon system;

[0021] Alternatively, transform the second plasmid into the Escherichia coli Nissle 1917 competent cells to obtain strains expressing the transposase; transform the third plasmid into the strains expressing the transposase, screen the strains that grow normally on a plate with phosphite as the sole phosphorus source, randomly pick colonies, detect green fluorescence, and judge the gene integration effect of the double plasmid transposon system.

[0022] This method uses the ptxD gene as the screening tag of the transposon system, and uses a medium with phosphite as the sole phosphorus source to screen strains with random gene integration of the transposon. At the same time, the output intensity of the reporter gene is used as an indicator of the expression level of the target gene to judge the integration effect; since the plasmid is removed in subsequent steps, the strains modified by this transposon system do not carry any resistance screening tags, thereby eliminating potential safety hazards in downstream applications and the metabolic burden brought by resistance genes.

[0023] In the third aspect of the present invention, the present invention provides the modified strains obtained by the above method without residual resistance screening tags.

[0024] In the fourth aspect of the present invention, the present invention provides the application of the above transposon system without residual resistance screening tags or the above method in modifying intestinal probiotics.

[0025] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For the principle of a method for editing strains using a transposon system with residual non-resistant screening tags according to an embodiment of the present invention;

[0027] Figure 2 For the production of green fluorescence of different transposon strains according to an embodiment of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions of the present invention are illustrated by specific specific examples below. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of each method step are only convenient tools for identifying each method step, rather than limiting the arrangement order of each method step or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope in which the present invention can be implemented.

[0029] To better understand the above technical solutions, the exemplary embodiments of the present invention will be described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0030] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market; the experiments involved are all conventional experimental methods unless otherwise specified.

[0031] Sources of materials used: Escherichia coli Nissle 1917, Escherichia coli strains TOP10 and DH5alpha-λ are commercially available, and TOP10 strain and DH5alpha-λ are used for vector construction. Phusion high-fidelity DNA polymerase and restriction endonucleases are purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kits, DNA purification kits, gel recovery kits and genomic DNA extraction kits are purchased from Hangzhou Bioer Technology Co., Ltd.

[0032] The composition of LB medium is: 10 g·L -1 peptone, 5 g·L -1 yeast extract, 5 g·L -1NaCl was made up to 1 L with double-distilled water and sterilized at 121 °C under a pressure of 0.1 Mpa for 20 min.

[0033] The composition of M9(-P) solid medium is as follows: 5×M9(-P) Salt (NH 4 Cl 2.5 g, NaCl 1.25 g, KH 2 PO 3 2 0.9 g, the pH was adjusted to 7.2, made up to 500 mL with double-distilled water, and filtered through a 0.22 μm filter for sterilization), 10×Glucose (200 g·L - 1 Glucose was made up to 1 L with double-distilled water and sterilized at 121 °C under a pressure of 0.1 Mpa for 20 min), 1000×trace elements (EDTA 50 g·L -1 , FeCl 3 ·6H 2 O 8.3 g·L -1 , ZnCl 2 0.84 g·L -1 , CuCl 2 ·2H 2 O 0.13 g·L -1 , CoCl 2 ·2H 2 O 0.1 g·L -1 , H 3 BO 3 0.1 g·L -1 , MnCl 2 ·4H 2 O 16 mg·L -1 , 1000×CaCl 2 (CaCl 2 0.55 g was dissolved in 45 mL of double-distilled water and then made up to 50 mL, and filtered through a 0.22 μm filter for sterilization), 1000×vitamins (thiamin-HCl 50 mg, biotin 50 mg, dissolved in 45 mL of double-distilled water and then made up to 50 mL, and filtered through a 0.22 μm filter for sterilization), 500×MgSO 4 (MgSO 4 6.02 g was dissolved in 45 mL of double-distilled water and then made up to 50 mL, and filtered through a 0.22 μm filter for sterilization), 15 g·L -1 agar.

[0034] The composition of M9(-P) liquid medium is as follows: 5×M9(-P) Salt (NH 4 Cl 2.5 g, NaCl 1.25 g, KH 2 PO 320.9 g, adjust the pH to 7.2, make up the volume to 500 mL with double-distilled water, filter and sterilize with a 0.22 μm filter), 10ⅹGlucose (200 g·L - 1 Glucose, make up the volume to 1 L with double-distilled water, sterilize at 121 °C under a pressure of 0.1 Mpa for 20 min), 1000ⅹ trace elements (EDTA 50 g·L -1 , FeCl 3 ·6H 2 O 8.3 g·L -1 , ZnCl 2 0.84 g·L -1 , CuCl 2 ·2H 2 O 0.13 g·L -1 , CoCl 2 ·2H 2 O 0.1 g·L -1 , H 3 BO 3 0.1 g·L -1 , MnCl 2 ·4H 2 O 16 mg·L -1 , filter and sterilize with a 0.22 μm filter), 1000ⅹ CaCl 2 (CaCl 2 0.55 g is dissolved in 45 mL of double-distilled water and then made up the volume to 50 mL, filter and sterilize with a 0.22 μm filter), 1000ⅹ vitamins (thiamin-HCl 50 mg, biotin 50 mg, dissolved in 45 mL of double-distilled water and then made up the volume to 50 mL, filter and sterilize with a 0.22 μm filter), 500ⅹ MgSO 4 (MgSO 4 6.02 g is dissolved in 45 mL of double-distilled water and then made up the volume to 50 mL, filter and sterilize with a 0.22 μm filter), after adding each component in proportion, make up the volume with double-distilled water.

[0035] The principle of the method for editing strains using a transposon system without residual resistance screening tags in this application is shown in Figure 1 :

[0036] Use a single-plasmid transposon system or a two-plasmid transposon system to randomly insert the reporter gene and the ptxD gene module P con -eGFP-ptxD into the host genome. Only the strains that have successfully integrated the ptxD gene can utilize phosphite, and the strains with successful transposition are screened on a plate with phosphite as the sole phosphorus source. The strains with successful transposition simultaneously express eGFP, and the fluorescence intensity of eGFP can reflect the gene expression intensity and be used as a basis for judging the gene transposition effect.

[0037] In the following examples, the nucleotide sequences of the plasmids are shown in Table 1.

[0038] Table 1 Nucleotide Sequences of Plasmids

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] The present invention will be described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0047] Example 1 Construction of a Single Plasmid Transposon System without Residual Resistance Screening Tags

[0048] The single plasmid transposon system consists of a plasmid, namely pKD3-Cm-P tetA -tnp*-Tn5-P con -eGFP-ptxD-Tn5 to complete transposition. P tetA The promoter initiates the expression of the transposase gene tnp to produce transposase, which recognizes the Tn5 sequence and randomly integrates the gene sequence P con -eGFP-ptxD between the two Tn5 sequences into the target host genome.

[0049] Specifically, the pKD3-Cm-Tn5 vector with the nucleotide sequence as shown in SEQ ID NO:1 was cut by NotⅠ-XhoⅠ, and the target band was recovered by a DNA purification kit to obtain a gel recovery product; it was ligated with the reporter gene and the ptxD gene module P con -eGFP-ptxD with the nucleotide sequence as shown in SEQ ID NO:2 after NotⅠ-XhoⅠ digestion, and then transferred into DH5alpha-λ competent cells, and the plasmid pKD3-Cm-Tn5-P 4 -eGFP-ptxD-Tn5 was extracted. The plasmid pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 con-eGFP-ptxD-Tn5 was cleaved by XbaⅠ and ligated with the transposase gene module P whose nucleotide sequence after XbaⅠ cleavage is shown as SEQ ID NO:5, thus obtaining the component plasmid of the single plasmid transposon system without residual resistance screening tag: pKD3-Cm-P tetA -tnp*, that is, the component plasmid of the single plasmid transposon system without residual resistance screening tag was obtained: pKD3-Cm-P tetA -tnp*-Tn5-P con -eGFP-ptxD-Tn5. pKD3-Cm-P tetA -tnp*-Tn5-P con -eGFP-ptxD-Tn5 was digested with XbaⅠ to prove that the P tetA -tnp* module was successfully ligated.

[0050] Example 2 Construction of a two-plasmid transposon system without residual resistance screening tag

[0051] The two-plasmid transposon system consists of two plasmids, namely pSC101-P tetA -tnp and pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 to complete transposition. After the plasmid pSC101-P tetA -tnp was transformed into the target host, the target host could express the transposase. Then, after the plasmid pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 was transformed into the target host that could express the transposase, the gene sequence P con -eGFP-ptxD between the Tn5 sequences was cut off by the transposase and randomly integrated into the target host genome.

[0052] Specifically, the pSC101 vector with the nucleotide sequence shown as SEQ ID NO:3 was cleaved by EcoRⅠ-SalⅠ and ligated with the transposase gene module P whose nucleotide sequence after EcoRⅠ-SalⅠ cleavage is shown as SEQ ID NO:4 tetA -tnp to obtain the plasmid pSC101-P tetA -tnp. The plasmid pSC101-P tetA -tnp and the plasmid pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 (constructed in the same way as in Example 1) constituted a two-plasmid transposon system without residual resistance screening tag. pSC101-P tetA -tnp was proved to be successfully ligated by digestion with EcoRⅠ and SalⅠ, and pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 was proved to be successfully ligated by digestion with NotⅠ and XhoⅠ.

[0053] Example 3 Editing Strain Using a Transposon System without Resistant Screening Tag Residue

[0054] Use the single - plasmid transposon system of Example 1 or the double - plasmid transposon system of Example 2 to randomly integrate the P con -eGFP-ptxD gene module into the genome of Escherichia coli Nissle 1917.

[0055] Specifically, transform the single - plasmid transposon system pKD3 - Cm - tnp*-Tn5 - P con -eGFP-ptxD-Tn5 into Escherichia coli Nissle 1917 competent cells, screen for strains that grow normally on plates with phosphite as the sole phosphorus source, randomly pick colonies, detect green fluorescence, and judge the gene integration effect of the single - plasmid transposon system. Or, transform the plasmid pSC101 - P tetA -tnp into the Escherichia coli Nissle 1917 competent cells to obtain strains expressing transposase; transform the pKD3 - Cm - Tn5 - P con -eGFP-ptxD-Tn5 into the strains expressing transposase, screen for strains that grow normally on plates with phosphite as the sole phosphorus source, randomly pick colonies, detect green fluorescence, and judge the gene integration effect of the double - plasmid transposon system.

[0056] Among them, after the plasmid of the single - plasmid transposon system or the plasmid of the double - plasmid transposon system is chemically transformed into Escherichia coli Nissle 1917 competent cells, it is activated at 37°C for 1 h and then spread on an M9(-P) solid medium with phosphite as the sole phosphorus source. The plate contains 8 - 9 mL of medium, and then it is cultured at 37°C for about 48 h. During this period, observe the colony growth situation. Bacteria that have not successfully transposed cannot grow. The culture condition of 37°C can remove the temperature - sensitive plasmid pSC101 - P tetA -tnp in the double - plasmid transposon system. The plasmids pKD3 - Cm - P tetA -tnp*-Tn5 - P con -eGFP-ptxD-Tn5 and the plasmid pKD3 - Cm - Tn5 - P con -eGFP-ptxD-Tn5 cannot replicate in Escherichia coli Nissle 1917. Therefore, there will be no plasmid residue in the cells of the transposed strains.

[0057] Transfer the single colonies growing on the M9(-P) plate to an M9(-P) liquid medium and culture at 37°C for 24 h. Use a microplate reader Biotek to detect the fluorescence intensity. Select an excitation wavelength of 480 nm and an emission wavelength of 520 nm to detect the fluorescence signal of green fluorescent protein, and judge the gene integration effect of the transposon system. The results are as followsFigure 2 As shown, the green fluorescence intensity of the control group is that of the unedited wild-type Escherichia coli Nissle 1917. Both the single-plasmid transposon system and the two-plasmid transposon system of the present application can efficiently edit the strain genome. Due to the randomness of the integration site and the number of integrated copies, the output intensity of the reporter gene of different transposon strains is different, which can meet different research needs and be used to construct different mutant libraries.

[0058] In summary, compared with the traditional transposon system, when editing strains using the transposon system without resistant screening label residues according to the embodiments of the present invention, most transpositions cannot be actually applied to subsequent intestinal treatment or production practice applications due to the limitation of the resistance label. Using this transposon system to edit strains can avoid the safety hazards and metabolic burdens brought by the residues of the resistance screening label, and is more competitive in actual production applications.

[0059] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0060] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A transposon system without resistance screening tag residue, characterized in that: The transposon system is a single-plasmid transposon system or a dual-plasmid transposon system; The single-plasmid transposon system includes a first plasmid that expresses a transposase and carries a reporter gene and a ptxD gene; The dual-plasmid transposon system includes a second plasmid expressing a transposase and a third plasmid carrying a reporter gene and a ptxD gene.

2. The transposon system without resistance screening tag residue according to claim 1, characterized in that: The reporter gene is the green fluorescent protein eGFP gene.

3. The transposon system without resistance screening tag residue according to claim 1, characterized in that: The transposon system is a non-replicating Tn5 transposon system.

4. The transposon system without resistance screening tag residue according to claim 1, characterized in that: The pKD3-Cm-Tn5 vector with the nucleotide sequence shown in SEQ ID NO: 1 was cut by NotⅠ-XhoⅠ, and the nucleotide sequence after NotⅠ-XhoⅠcut was the reporter gene and ptxD gene module P shown in SEQ ID NO: 2 con -eGFP-ptxD was connected to obtain pKD3-Cm-Tn5-P con -eGFP-ptxD-Tn5 plasmid, which is the third plasmid; pKD3-Cm-Tn5-P con The -eGFP-ptxD-Tn5 plasmid was cut by XbaⅠ, and the nucleotide sequence after XbaⅠ cutting was as shown in SEQ ID NO:

5. tetA -tnp* ligation to obtain pKD3-Cm-P tetA -tnp*-Tn5-P con -eGFP-ptxD-Tn5 plasmid, which is the first plasmid.

5. The transposon system without resistance screening tag residue according to claim 1, characterized in that: The pSC101 vector with the nucleotide sequence shown in SEQ ID NO:3 was cut by EcoRI-SalI, and the nucleotide sequence after EcoRI-SalI cutting was the transposase gene module P shown in SEQ ID NO:4 tetA -tnp to obtain pSC101-tnp plasmid, which is the first plasmid.

6. A method for editing a strain using the transposon system without resistance screening tag residues according to any one of claims 1 to 5, characterized in that: include: randomly integrating the transposon system into the genome of the target host; The target host is screened for colonies that can grow normally on a plate with phosphite as the only phosphorus source to obtain a strain with successful gene integration. At the same time, the output intensity of the reporter gene is used as an indicator of the expression amount of the target gene to judge the integration effect.

7. The method according to claim 6, characterized in that The target host is Escherichia coli Nissle 1917.

8. The method according to claim 6, characterized in that Transforming the first plasmid into competent Escherichia coli Nissle1917 cells, screening strains that grow normally on a plate with phosphite as the only phosphorus source, randomly picking colonies, detecting green fluorescence, and judging the gene integration effect of the single-plasmid transposon system; Alternatively, the second plasmid is transformed into the Escherichia coli Nissle 1917 competent cells to obtain a strain expressing the transposase; the third plasmid is transformed into the strain expressing the transposase, and the strains that grow normally are screened on a plate with phosphite as the only phosphorus source, and colonies are randomly picked to detect green fluorescence to determine the gene integration effect of the dual-plasmid transposon system.

9. The method according to any one of claims 6 to 8 obtains a transformed strain having no resistance screening tag residue.

10. Use of the transposon system without resistance screening tag residues as described in any one of claims 1 to 5 or the method as described in any one of claims 6 to 8 in transforming intestinal probiotics.

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