Copy number regulation and control method based on 2mu plasmid and application of copy number regulation and control method

Abstract

The invention relates to the technical field of synthetic biology, and discloses a copy number regulation and control system based on a 2 mu plasmid, the copy number regulation and control system comprises a plasmid with a controllable copy number and an expression system thereof, and the regulation and control method comprises the following steps: S1, inserting an endogenous protein sequence driven and expressed by promoters with different intensities into a genome of a saccharomyces cerevisiae cell or the plasmid; s2, the saccharomyces cerevisiae cells or the plasmids express endogenous proteins with different intensities; s3, transferring a 2 mu plasmid without the endogenous protein sequence; and S4, finishing the copy number regulation and control of the 2 mu plasmid. According to the method, the copy number of the plasmid is changed and the range of the copy number is expanded by artificially modifying the expression quantity of the replication isolated protein of the endogenous 2mu plasmid, and an antibiotic or an inducer does not need to be added, so that the culture cost is greatly reduced, and the growth speed of the strain is almost not influenced.

Description

Technical Field

[0001] The present application relates to the field of synthetic biotechnology, and mainly to a copy number regulation method based on a 2μ plasmid and its application. Background Art

[0002] Plasmids are small, circular DNA molecules in bacteria and other cells. They are double-stranded DNA fragments that replicate independently outside the chromosome. In synthetic biology, plasmids are used as vectors and are widely used in DNA cloning, gene editing, gene recombination expression, and construction of gene loops. The copy number of plasmids, that is, the number of plasmids in a cell, is crucial to the efficiency and stability of gene expression. Therefore, the copy number of plasmids is the primary issue that needs to be considered in the application of plasmids. In prokaryotes such as Escherichia coli, several systems that can regulate the copy number of plasmids have been initially established. The copy number of plasmids can be regulated by different mechanisms, such as using different replication origins and replication control proteins. These systems can adjust the copy number of plasmids as needed to optimize gene expression or avoid potential toxic effects.

[0003] In prokaryotes such as Escherichia coli, the regulation mechanism of plasmid copy number is relatively mature, and researchers have developed several different systems to control plasmid copy number. Most strategies can only statically regulate plasmid copy number, that is, the copy number is fixed, and static regulation of plasmid copy number is achieved by changing the origin of replication sequence (Ori) or replication regulatory elements through mutation, overexpression of specific genes or integration of specific transcription factors. For example, in pCEs plasmids, the copy number is statically regulated by adjusting the mutation of ParB protein-related replication process in other regions involved in regulating DNA replication process in addition to the main replication origin, which increases the plasmid copy number by nearly 10 times. However, very few studies have achieved dynamic regulation of copy number. So far, only pSC101 and ColE1 plasmids for Escherichia coli have achieved dynamic regulation of copy number, controlling the expression of replication proteins through inducible promoters or riboswitches, thereby achieving dynamic regulation of plasmid copy number. However, there are some challenges in directly applying these strategies in prokaryotic systems to eukaryotic cells, such as yeast cells. Since eukaryotic cells have different cell structures and gene expression regulation mechanisms, such as the presence of nuclear membranes, complex chromatin structures, and different transcription and translation regulation pathways, it is impossible to apply the plasmid copy number regulation mechanism in prokaryotes to eukaryotic cells. Currently, all reports use promoter truncation engineering or different concentrations of antibiotics to achieve static regulation of eukaryotic cell plasmid copy number. For example, in Saccharomyces cerevisiae, by truncating the promoter of the selection marker and adjusting the concentration of the antibiotic G418, the plasmid reaches 5-100 copies, achieving static regulation of copy number. However, this method requires the host to be cultured in a nutritional deficiency or a high concentration of antibiotics, which creates a metabolic burden on the host and seriously interferes with applications such as metabolic optimization and the study of multi-copy genes.

[0004] In summary, new strategies and technologies need to be developed to achieve the regulation of plasmid copy number in eukaryotic cells. Summary of the invention

[0005] In response to the technical problems in the prior art, the present application proposes a copy number regulation method based on a 2μ plasmid and its application.

[0006] According to one aspect of the present invention, a copy number control method based on a 2μ plasmid is proposed, comprising the following steps:

[0007] S1, inserting endogenous protein sequences driven by promoters of different strengths into the genome of Saccharomyces cerevisiae cells or on plasmids;

[0008] S2, the Saccharomyces cerevisiae cell or the plasmid expresses endogenous proteins of different intensities;

[0009] S3, transfer into a 2μ plasmid lacking the endogenous protein sequence;

[0010] S4. Complete the copy number regulation of the 2μ plasmid.

[0011] Preferably, in said S1, the endogenous protein sequences include FLP gene, REP1 gene, REP2 gene and RAF1 gene.

[0012] Preferably, in S1, the insertion site of the promoter is the front end of the endogenous protein sequence.

[0013] Preferably, in said S2, the endogenous proteins include Flp protein, Rep1 protein, Rep2 protein and Raf protein.

[0014] In a specific embodiment, the natural 2μ plasmid in the Saccharomyces cerevisiae cell encodes four proteins, and its replication depends on the specific recombination of FRT mediated by the FLP site-specific recombinase to achieve rolling circle replication; and separation is through the formation of a complex between the REP1 / REP2 protein and the specific repeat sequence of STB, which adheres to the chromosome and separates stably with it; RAF1 plays a regulatory role in negative feedback inhibition. Therefore, the copy number of the 2μ plasmid is regulated by the expression of FLP, REP1, REP2 and RAF1. The four proteins are expressed by their own promoters, and the strength of the promoter determines their expression level, which ultimately determines the copy number of the plasmid. On this basis, FLP, REP1, REP2 and RAF1 are modified by promoter engineering, so that the expression of FLP, REP1, REP2 and RAF1 is artificially controlled.

[0015] In a specific embodiment, the promoter includes a low-strength promoter CYC1, a high-strength promoter TDH3 and an inducible high-strength promoter Gals, the low-strength promoter CYC1 sequence is shown in SEQ ID NO.1, the high-strength promoter TDH3 sequence is shown in SEQ ID NO.2, and the inducible high-strength promoter Gals sequence is shown in SEQ ID NO.3.

[0016] Preferably, no external antibiotics or inducers are required during the entire regulation process of the 2μ plasmid-based copy number regulation method.

[0017] Traditional methods for static regulation of copy number (e.g., using promoter truncation engineering or different concentrations of antibiotics to achieve static regulation of plasmid copy number) require the host to be cultured in a nutritionally deficient or high concentration of antibiotics, which easily increases the metabolic burden of the host and causes serious interference with applications such as metabolic optimization and research on multi-copy genes. However, the present invention artificially modifies the expression of replication and separation proteins of endogenous 2μ plasmids, without the need for external antibiotics or inducers, and without screening pressure, greatly reducing the culture cost, and having almost no effect on the growth rate of the strain.

[0018] Preferably, the copy number detection method includes quantifying the copy number by qPCR and inserting mCherry into the plasmid to detect the copy number by flow cytometry.

[0019] qPCR detection has the advantages of high sensitivity, strong specificity, accurate quantification, simple operation and good repeatability. mCherry detection has the advantages of strong photostability and low cytotoxicity. Combining the two for copy number detection helps to obtain more reliable experimental results.

[0020] According to the second aspect of the present invention, a plasmid with controllable copy number is proposed, which is obtained by a copy number regulation method based on a 2μ plasmid, and includes a 2μori replicon, an IR inverted repeat sequence, an auxiliary quantitative gene fluorescent protein mCherry and a plasmid screening marker HIS3.

[0021] According to the third aspect of the present invention, a copy number regulation method based on 2μ plasmid is proposed for application in gene expression regulation, clone screening, vaccine development, metabolic engineering and gene therapy.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) This application achieves static and dynamic controllable regulation of copy number in eukaryotic cells by modifying 2μ elements, thereby rapidly amplifying and extracting target DNA fragments, optimizing metabolic modules, overexpressing vaccine proteins, and precisely controlling gene expression in Saccharomyces cerevisiae and even eukaryotic cells;

[0024] (2) The existing plasmid copy number of Saccharomyces cerevisiae is low, and the constructed plasmid cannot be directly extracted. The present application assembles DNA elements in Saccharomyces cerevisiae and amplifies the copy number, and directly extracts the plasmid, which greatly simplifies the DNA assembly and amplification process;

[0025] (3) The plasmid regulatory system of the present application can also be used to study multi-copy genes, such as rDNA, tRNA and histone genes, by regulating their copy numbers in cells to further explore their diversity and their potential roles in evolution, disease and biological functions;

[0026] (4) The replication and separation method of the 2μ plasmid of the present application is unique and relatively independent, and can rely on its own coupling system to ensure stability. It is hoped that a plasmid system can be constructed in eukaryotic cells through transformation to achieve stable, continuous and efficient expression of the target gene;

[0027] (5) The plasmid regulation method of the present application does not require the addition of antibiotics or inducers, which greatly reduces the culture cost and has almost no effect on the growth rate of the strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated into and constitute a part of this specification. The accompanying drawings illustrate the embodiments and are used together with the description to explain the principles of the present invention. It will be easy to recognize other embodiments and many expected advantages of the embodiments because they become better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale with each other. The same reference numerals refer to corresponding similar parts.

[0029] Figure 1 A schematic diagram of a copy number control method based on a 2μ plasmid according to an embodiment of the present invention is shown;

[0030] Figure 2 A diagram showing the mCherry detection result according to a specific embodiment of the present invention is shown;

[0031] Figure 3 A graph showing the results of qPCR assay according to a specific embodiment of the present invention is shown. DETAILED DESCRIPTION

[0032] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0033] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0034] Figure 1 A schematic flow chart of a copy number control method based on a 2μ plasmid according to an embodiment of the present invention is shown, as shown in FIG. Figure 1 As shown, the specific steps include:

[0035] S1, inserting FLP / REP1 / REP2 / RAF1 endogenous protein sequences of different strength promoters into the genome of Saccharomyces cerevisiae cells or on plasmids, the promoters include low strength promoter CYC1, high strength promoter TDH3 and inducible medium strength promoter Gals;

[0036] S2, Saccharomyces cerevisiae cells or plasmids expressing Flp / Rep1 / Rep2 / Raf endogenous proteins at different intensities;

[0037] S3, transfer of 2μ plasmid lacking the endogenous protein sequences of Flp / Rep1 / Rep2 / Raf;

[0038] S4. Complete the regulation of 2μ plasmid copy number. The 2μ plasmid copy number changes accordingly with the increase of Flp / Rep1 / Rep2 / Raf endogenous protein expression.

[0039] In a specific embodiment, the constructed plasmids are transformed into yeast strains with 2μ endogenous proteins of different strength promoters, namely CYC1-FLP-HA strain, TDH3-FLP-HA strain and Gals-FLP-HA strain (Gals-FLP-HA strain includes Gal-glu-FLP-HA strain and Gal-gal-FLP-HA strain), wherein the carbon source of CYC1-FLP-HA strain, TDH3-FLP-HA strain and Gal-glu-FLP-HA strain is glucose, and the carbon source of Gal-gal-FLP-HA strain is galactose.

[0040] Each strain was cultured in SC-His medium (30°C, 220 rpm) until the OD value reached the stationary phase. 600 Afterwards, observe the color of the bacteria. The depth of the pink-purple color of the colonies is proportional to the number of copies. Figure 2 As shown, the color of the TDH3-FLP-HA strain is darker than that of the CYC1-FLP-HA strain, the Gal-glu-FLP-HA strain, and the Gal-gal-FLP-HA strain. As the promoter strength increases, the strain color becomes darker. This proves that as the promoter strength increases, the plasmid copy number also gradually increases.

[0041] In addition, the plasmid copy number was quantitatively determined by qPCR, and the measurement process was as follows: 1) the bacterial cells were collected after centrifugation of the strain; 2) yeast DNA was extracted using the phenol chloroform isoamyl alcohol method; 3) qPCR SYBR Green Master Mix (No Rox) (Yisheng Biotechnology) was used for copy number quantitative detection. Among them, the detection primer used the AMP gene on the plasmid, the upstream primer sequence was shown in SEQ ID NO.4, the downstream primer sequence was shown in SEQ ID NO.5, and the internal reference primer used the ALG9 gene of the Saccharomyces cerevisiae genome, the upstream primer sequence was shown in SEQ ID NO.6, and the downstream primer sequence was shown in SEQ ID NO.7. The test results are shown in Figure 3 As shown, from Figure 3 It can be seen that with the increase of promoter strength, the number of plasmid copies is also gradually increasing. The Gal promoter is an inducible promoter with low promoter strength in glucose. The number of copies in each genome of the Gal-glu-FLP-HA strain is only about 5, which is lower than the 10 of the CYC1-FLP-HA strain. The Gal-gal-FLP-HA strain has a higher promoter strength in galactose, with a copy number of nearly 30, second only to the 40 of the TDH3-FLP-HA strain.

[0042] The above results prove that inserting promoters of different strengths into the Saccharomyces cerevisiae genome can effectively affect the plasmid copy number, and the copy number increases with the increase in the expression level of endogenous protein. The above technical solution can achieve dynamic regulation of the plasmid copy number.

[0043] The present invention artificially modifies the replication and separation protein expression of the endogenous 2μ plasmid, thereby changing the copy number of the plasmid and expanding the range of the copy number. This method does not require the addition of antibiotics or inducers, greatly reduces the culture cost, and has almost no effect on the growth rate of the strain.

[0044] The sequences of the promoter and primers involved in the examples are shown in Table 1.

[0045] Table 1 Promoter and primer sequences used in this application

[0046]

[0047]

[0048] The above describes the specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A copy number control method based on 2μ plasmid, characterized in that: The following steps are involved: S1, inserting endogenous protein sequences driven by promoters of different strengths into the genome of Saccharomyces cerevisiae cells or on plasmids; S2, the Saccharomyces cerevisiae cell or the plasmid expresses endogenous proteins of different intensities; S3, transfer into a 2μ plasmid lacking the endogenous protein sequence; S4. Complete the copy number regulation of the 2μ plasmid.

2. The copy number control method based on 2μ plasmid according to claim 1, characterized in that: In the S1, the endogenous protein sequences include FLP gene, REP1 gene, REP2 gene and RAF1 gene.

3. The copy number control method based on 2μ plasmid according to claim 1, characterized in that: In the S1, the insertion site of the promoter is the front end of the endogenous protein sequence.

4. The copy number control method based on 2μ plasmid according to claim 1, characterized in that: In the S2, the endogenous proteins include Flp protein, Rep1 protein, Rep2 protein and Raf protein.

5. The copy number control method based on 2μ plasmid according to claim 1, characterized in that: No external antibiotics or inducers are required in the entire regulation process of the copy number regulation method based on the 2μ plasmid.

6. The copy number control method based on 2μ plasmid according to claim 1, characterized in that: The copy number detection method includes quantifying the copy number by qPCR and inserting mCherry into the plasmid to detect the copy number by flow cytometry.

7. A plasmid with controllable copy number, characterized in that: The method for controlling the copy number of a 2μ plasmid according to any one of claims 1 to 6 is used, wherein the plasmid with controllable copy number includes a 2μori replicon, an IR inverted repeat sequence, an auxiliary quantitative gene fluorescent protein mCherry, and a plasmid screening marker HIS3.

8. Application of the 2μ plasmid-based copy number control method according to any one of claims 1 to 6 in gene expression control, clone screening, vaccine development, metabolic engineering and gene therapy.

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