Application of plasmid elimination module based on cascade reaction

Abstract

The invention relates to the field of gene engineering, in particular to application of a plasmid elimination module based on cascade reaction, and the plasmid elimination module is used for selective elimination of a specified object plasmid or synchronous elimination of several specified object plasmids. The method is high in elimination efficiency, elimination objects can be freely set according to needs, and selective or synchronous elimination of various plasmids can be realized.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to the application of a plasmid elimination module based on cascade reaction. Background Art

[0002] Free plasmids are a universal platform for genetic manipulation of animal, plant and microbial cells, and can be used for purposes such as genome editing, heterologous protein expression, and gene circuit construction. However, when genome editing or gene circuit plasmids are used to achieve the purpose of genome editing or gene circuit design, the genome editing tools or gene circuits carried on the plasmids become unstable factors in the cell genome. At this time, the plasmids need to be eliminated to obtain plasmid-free cells. The current plasmid elimination technologies are mainly divided into three types, namely, temperature-sensitive replicon plasmid elimination, sacB plasmid elimination, and CRISPR protein-related plasmid elimination technology.

[0003] Thermosensitive replicon plasmid elimination efficiency is low, and it is often used for low-copy plasmid elimination, but it is not effective for medium- and high-copy plasmid elimination. In addition, the same replicon in the same cell can generally only serve one plasmid at the same time. If the thermosensitive replicon is used to serve multiple plasmids, the spontaneous loss of plasmids may be aggravated, which is not conducive to the development of related experiments or production work;

[0004] Plasmid elimination based on the sacB gene can serve multiple plasmids at the same time, but when multiple plasmids exist in the cell, the plasmid elimination method based on the sacB gene cannot accurately eliminate a specific type of plasmid, and its tolerance to mutation is weak and it is easy to fail;

[0005] CRISPR protein-related plasmid elimination. Plasmid elimination technology based on CRISPR protein can eliminate multiple plasmids at the same time and freely select the type of plasmid that needs to be eliminated. However, plasmid elimination technology based on CRISPR protein has the risk of CRISPR protein gene mutation failure or CRISPR protein cutting off-target, and the gene sequence of CRISPR-related protein elements is long, which is not conducive to use in chassis microorganisms that are difficult to transfer large plasmids. When there is no natural CRISPR protein available in the chassis cells, additional plasmids with CRISPR proteins need to be transferred, and the failure rate of self-targeted elimination of plasmids with CRISPR proteins is high. Summary of the invention

[0006] The present invention proposes a plasmid elimination technology based on antisense RNA, designs the application of a plasmid elimination module, which can promote the loss of free plasmids in cells, can efficiently achieve the selective elimination or synchronous elimination of multiple free plasmids, can be applied to the selective elimination or synchronous elimination of different types (copy numbers) of free plasmids in eukaryotic and prokaryotic cells, and has better tolerance to sequence mutations than the plasmid elimination technology based on CRISPR protein. The gene sequence length of the related protein element is shorter than that of CRISPR, and it is still applicable to chassis cells that are difficult to transfer large plasmids.

[0007] Antisense RNA designed for key metabolic genes can reduce the expression of key metabolic genes, thereby affecting the cell growth rate. During cell amplification and passage, plasmids are randomly distributed to offspring. The number of offspring cells containing the plasmids of this elimination module will decrease, while the number of offspring cells without the plasmids of this elimination module will increase due to rapid growth, thereby achieving the purpose of enriching plasmid-free cells and achieving plasmid elimination.

[0008] The plasmid elimination module designed by the present invention can be deployed in the cell where the plasmid to be eliminated is located, except for the antisense RNA expression region which must be deployed on the plasmid to be eliminated. After the deployment is completed, the plasmid elimination module designed by the present invention can efficiently achieve plasmid elimination within the deployment range.

[0009] The significance of the present invention lies in proposing a universal plasmid elimination module construction idea that is convenient, usable, programmable, widely adaptable, highly stable, and requires a shorter sequence. Under the condition that the preconditions are met, the plasmid elimination module can be used for the elimination of constructed plasmids after the construction of genetically engineered bacteria and the elimination of artificially constructed plasmids.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] An application of a plasmid elimination module based on cascade reaction is used for the selective elimination of a designated target plasmid or the selective simultaneous elimination of several designated target plasmids.

[0012] According to an embodiment of the present invention, the plasmid elimination module includes three types of independent expression regions, one of which is an antisense RNA expression region, which includes antisense RNA, a promoter and a terminator of the antisense RNA; the antisense RNA is the antisense RNA of the key metabolic gene mRNA, which can bind to the key metabolic gene mRNA to hinder the translation of the key metabolic gene mRNA; one type of region is an antisense neutralizing RNA expression region, which includes antisense neutralizing RNA, an inducible promoter and a terminator of the antisense neutralizing RNA; the last type of region is a transcription factor region, which is composed of a constitutive promoter of the transcription factor fragment, which includes an RNA polymerase binding site and a transcription start site, a ribosome binding site, a transcription factor translation region, and a terminator; the antisense neutralizing RNA is complementary to the antisense RNA sequence, can bind to the antisense RNA so that it cannot bind to the key metabolic gene mRNA and inhibit the antisense RNA from producing a translation-hindering RNA fragment on the key metabolic gene mRNA.

[0013] According to an embodiment of the present invention, the transcription factor is a protein that can induce transcription of genes downstream of the binding site after binding to a specific compound; or a protein that can induce transcription of downstream genes before binding to a specific compound, but cannot induce transcription of genes downstream of the binding site after binding to the specific compound.

[0014] According to an embodiment of the present invention, the transcription factor is an arabinose transcription factor.

[0015] According to an embodiment of the present invention, the constitutive promoter of the transcription factor fragment is the constitutive promoter of the arabinose transcription factor.

[0016] According to an embodiment of the present invention, the antisense RNA promoter includes J23119 promoter, T7, tet, trc, trp, tac and constitutive promoters related to chloramphenicol, kanamycin, and penicillin resistance genes commonly used in glutamicum antibiotics, or TEF1 promoter, and the antisense RNA terminator includes Cyc1 terminator, rrnB T1 terminator or rrnB T2 terminator.

[0017] According to an embodiment of the present invention, the antisense neutralizing RNA inducible promoter includes a GAL1 promoter and an arabinose promoter, and the antisense RNA terminator includes a Cyc1 terminator, an rrnB T1 terminator or a T7 terminator.

[0018] According to an embodiment of the present invention, the plasmid elimination module is mounted in the plasmid to be eliminated.

[0019] According to an embodiment of the present invention, the plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and yeast 2μ plasmid.

[0020] According to an embodiment of the present invention, the key metabolic genes are TCA cycle related genes, protein synthesis related genes, aerobic respiration related genes, anaerobic respiration related genes, lipid synthesis related genes, cell wall synthesis related genes, sugar transport pathway related genes, amino acid transport pathway related genes, ATP synthesis related genes, cytoskeleton synthesis related genes, NADPH / NADP + Synthesis-related genes, NADH / NAD + Synthesis-related genes, vitamin synthesis-related genes, one-carbon transport system-related genes, carbon dioxide fixation-related genes, DNA / RNA synthesis-related genes, amino acid synthesis-related genes, ribose synthesis-related genes, ribosome synthesis-related genes, ribosome assembly-related genes, aminoacyl-tRNA synthetase-related genes and isoenzyme genes of the above genes.

[0021] According to an embodiment of the present invention, the plasmid elimination module eliminates all microorganisms, animals or plant cells that have stable free plasmids, free plasmids that have a certain probability of not being distributed to daughter cells, and free plasmids that will not cause cell death or proliferation failure after elimination.

[0022] The design idea of ​​the plasmid elimination module proposed in the present invention is the core of the invention. The design achieves plasmid elimination by inducing an increase in the difference in growth rate between cells containing plasmids and cells without plasmids. It can be applied to all plasmids that have spontaneous loss but low spontaneous loss efficiency. The characteristics of the cascade reaction make it easy to form a multiple signal system for sequential biological behavior control.

[0023] To facilitate those skilled in the art to understand the present invention, the main terms are explained as follows:

[0024] Gene circuit: A combination of DNA sequences that combines specific gene elements such as promoters, terminators, protein translation regions, and specific protein binding regions according to certain rules and arranges them at single sites or multiple sites to achieve specific functions.

[0025] Convenient and usable: The module can be stored in the form of PCR products / plasmids, and can be inserted into extracellular or intracellular plasmids through Gibson assembly, Golden Gate cloning, enzyme ligation, genome editing, etc.

[0026] Programmable: The module can be used regardless of the number of plasmid copies and can be selectively eliminated in cells containing multiple plasmids by combining different regulatory proteins.

[0027] Wide adaptability: The module can be used across species by means of codon optimization / replacement of related control elements.

[0028] Strong stability: Even if there is a certain sequence deviation between the antisense RNA and the target mRNA, it does not affect the function of the module proposed by the present invention.

[0029] The required sequence is shorter: when using cell background regulatory factors, the total length of the plasmid elimination module sequence proposed in the present invention can usually be less than 1kbp, or even less than 100bp. When using self-contained regulatory factors, the total length of the plasmid elimination module sequence proposed in the present invention usually does not exceed 3kbp.

[0030] Constructing plasmid: mainly refers to the tool plasmid that is equipped with the necessary functional proteins for genome editing to assist in achieving the purpose of genome editing in genetically engineered cells. This type of plasmid usually needs to be eliminated after the genome editing of the engineered cells is completed to avoid introducing or causing additional mutations in the application of engineered cells.

[0031] Artificially constructed plasmids: mainly refers to plasmids that are artificially constructed to express homologous / heterologous proteins or some functional gene circuits without involving genome editing.

[0032] Antisense neutralizing RNA: It is complementary to the antisense RNA sequence and can bind to the antisense RNA so that it cannot bind to the mRNA of key metabolic genes and inhibit the antisense RNA from hindering the translation of the mRNA of key metabolic genes.

[0033] Timing: Controllable time difference.

[0034] Free plasmid: can autonomously assist or replicate with the genome in the cell, can be distributed to daughter cells as the cell proliferates, and can stabilize linear or circular double-stranded DNA outside the free genome.

[0035] Plasmid backbone: DNA sequence that carries the plasmid replicon and plasmid maintenance genes.

[0036] Constitutive promoter: A specific DNA sequence that can be recognized by the Escherichia coli sigma factor and recruit RNA polymerase subunits for downstream transcription.

[0037] The following are some examples:

[0038] E. coli related sequences:

[0039] The complete sequence of the interference region of the Escherichia coli acnB gene:

[0040] ACTATGACAATGAGAGCGAGGAGAACCGTCGTGCTAGAAGAATACCGTAAGCACGTAGCTGAGCGTGCCGCTGAGGGGATTGCGCCCAAACCCCTGGATGCAAACCAAATGGCCGCACTTGTAGAGCTGCTGAAAAACCCGCCCGCGGGCGAAGAAGAATTCCTGTTAGATCT (omitted here, see the sequence list for details)

[0041] GGCGCTGATTGGCAAACTGCCGACGCCGGAAGAGTACCAGACCTACGTGGCGCAAGTA

[0042] GATAAAACTGCCGTTGATACTTATCGTTATCTGAACTTCAACCAGCTTTCTCAGTACACC GAAAAAGCCGATGGGGTGATTTTCCAGACTGCGGTTTAA (SEQ ID NO: 1)

[0043] pET28a plasmid full sequence:

[0044] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT (omitted here, see the sequence list for details)

[0045] TAACTTTAAGAAGGAGATATACCATGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC

[0046] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGG GTTTTTTCTGAAAGGAGGAACTATATCCGGAT (SEQ ID NO: 2)

[0047] Arabinose promoter sequence (arrow indicates the direction of transcription initiation):

[0048] AAGAAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAA AAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ IDNO: 3)

[0049] T7 terminator sequence (arrow indicates the direction of transcription termination):

[0050] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG (SEQ ID NO: 4)

[0051] Arabinose transcription factor expression frame (including promoter, CDS region, terminator):

[0052] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACCTGCATCGATTTATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAG (omitted here, see the sequence list for details)

[0053] GCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAAACCAATTGTCCATATTGCATCAGA

[0054] CATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTT

[0055] ATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCT (SEQID NO: 5)

[0056] J23119 Promoter:

[0057] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC→(SEQ ID NO: 6)

[0058] rrnB T1 terminator:

[0059] CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC (SEQ ID NO: 7)

[0060] Corynebacterium glutamicum related sequences:

[0061] The complete sequence of the CDS region of the acn gene of Corynebacterium glutamicum:

[0062] TTGGAGCTCACTGTGACTGAAAGCAAGAACTCCTTCAATGCTAAGAGCACCCTTGAAGTTGGCGACAAGTCCTATGACTACTTCGCCCTCTCTGCAGTGCCTGGCATGGAGAAGCTGCCGTACTCCCTCAAGGTTCTCGGAGAGAACCTTCTTCGTACCGAAGACGGCGCAAAC (omitted here, see the sequence list for details)

[0063] GAGAACGGCGACGTCGTCGAGTTCGACGCAGTTGTCCGCATCGACACCCCAGGTGAGG

[0064] CTGACTACTACCGCCACGGCGGCATCCTGCAGTACGTGCTGCGTCAGATGGCTGCTTCTT CTAAGTAA (SEQ ID NO: 8)

[0065] Corynebacterium glutamicum pEC-XK99E plasmid sequence:

[0066] GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGA (omitted here, see the sequence list for details)

[0067] GCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACAT

[0068] CATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTAT AATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATG (SEQ ID NO: 9)

[0069] Arabinose promoter sequence (arrow indicates the direction of transcription initiation):

[0070] AAGAAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAA AAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ IDNO:10)

[0071] T7 terminator sequence (arrow indicates the direction of transcription termination):

[0072] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG (SEQ ID NO: 11)

[0073] Arabinose transcription factor expression frame (including promoter, CDS region, terminator):

[0074] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACCTGCATCGATTTATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAG (omitted here, see the sequence list for details)

[0075] CATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTT

[0076] ATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAA GTGTCT (SEQID NO: 12)

[0077] Trc Promoter:

[0078] TTGACAATTAATCATCCGGCTCGTATAATG→(SEQ ID NO: 13)

[0079] rrnB T2 terminator:

[0080] →AGAAGGCCATCCTGACGGATGGCCTTTT (SEQ ID NO: 14)

[0081] Saccharomyces cerevisiae related sequences:

[0082] The complete sequence of the CDS region of the Saccharomyces cerevisiae ACO1 / GLU1 gene:

[0083] ATGCTGTCTGCACGTTCTGCCATCAAGAGACCCATTGTTCGTGGTCTTGCGACAGTCTCCAACTTGACTAGAGATTCAAAAGTCAACCAAAACTTATTAGAAGATCATTCTTTTATTAACTACAAGCAGAATGTGGAAACGCTGGATATCGTAAGAAAAAGATTAAACAGGCCATT (omitted here, see the sequence list for details)

[0084] GAGTTCATCCAAAGAATGGTAAGCCATGGGATGCTGTGTTGACCCATACTTTCAACGATG

[0085] AGCAAATTGAATGGTTCAAATATGGTTCTGCCTTAAATAAAATTAAGGCCGATGAGAAGA AATAA (SEQ ID NO: 15)

[0086] Saccharomyces cerevisiae 2U shuttle plasmid sequence:

[0087] CAACTTTGTATAGAAAAGTTGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTT (omitted here, see the sequence table for details)

[0088] GCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGG

[0089] CGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCG CCGCTACAGGGCGCGTGGGGATCGATCCACTAGT (SEQ ID NO: 16)

[0090] Saccharomyces cerevisiae neomycin sulfate resistance gene sequence (including promoter, expression cassette, terminator):

[0091] GTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTG(SEQ IDNO:17)

[0092] Shuttle vector E. coli replicon sequence:

[0093] (SEQ ID NO: 18)

[0094] Shuttle vector ampicillin resistance gene expression cassette (including promoter, expression cassette, terminator):

[0095]

[0096] Sequence of the inducible promoter GAL1 of Saccharomyces cerevisiae:

[0097] ACGGATTAGAGCCGCCGAGCGACGAGCGAGCGGAGCGCAGCAGCGCCGAAGGAAGACTCTCCTCCGTGCGCTCCTCGTCTCGTCTTCACCGGTCGTCGTCCTGAAACGAGATGTGTGCCTCGCGCCGCACTGCTCCGAACAATAATACAATAAAGATTCTACAATACTAGCTATGGTTATGGTTATGAAGAGAAAAATGGCAGTACCTGGCCCCAAACCTCAAATGAACGAATCAATTAACAACCATAGGATGATGATAATGCGATTAGTTAGCTTATTCTATTTCTGGGGTAATTAATCAGCGAAGCGATGATGATTTTGATCTATTACAGATATATAATGCAAAACTGCATAACCACTTAACTAACTAATATAATACTTTCAACATTTTCGTGTTGATTACTTCTTATCAAATGTAATACAAAAATTGTAATATATACCTATACTACTACTACTTAACGTCAAGGAGACC(SEQ ID NO: 20)

[0098] Sequence of the terminator CYC1 of yeast:

[0099] ATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCT(SEQ ID NO: 21)

[0100] Sequence of the constitutive promoter TEF1 of Saccharomyces cerevisiae:

[0101] CCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAA AAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG(SEQ ID NO:22)

[0102] The beneficial effects of the present invention are as follows: the present invention designs a modular plasmid elimination technology based on antisense RNA, the elements required for constructing the plasmid elimination module are simple and easy to obtain, the plasmid module elimination efficiency is high, the elimination object can be freely set according to the needs, and the selective or synchronous elimination of multiple plasmids can be achieved. And the present invention has a wide range of applications, and the technology can be applied to both eukaryotic and prokaryotic organisms. The genes related to the central metabolic pathway can all be combined with antisense RNA for plasmid elimination. The antisense RNA of the metabolic-related genes such as amino acids, nucleic acids, lipid synthesis pathways, transcription and translation, DNA replication, cell walls, cell membranes, endoplasmic reticulum, etc. outside the central metabolic pathway can also be applied to this technology. If the elimination effect is not good, the elimination effect can be enhanced by increasing the types of antisense RNAs, extending the length of antisense RNAs, and other strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 This is the interference result diagram of Example 4.

[0104] Figure 2 This is the interference result diagram of Example 5.

[0105] Figure 3 This is the interference result diagram of Example 6.

[0106] Figure 4 It is a schematic diagram of the principle of the present invention. DETAILED DESCRIPTION

[0107] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0108] The purpose of setting up antisense neutralizing RNA is that the antisense neutralizing RNA sequence can be complementary to the antisense RNA sequence, can bind to the antisense RNA so that it cannot bind to the mRNA of the key metabolic gene and inhibit the obstruction of the antisense RNA on the translation of the mRNA of the key metabolic gene, so that the plasmid can be stably present in the cell when there is no need to eliminate the plasmid.

[0109] The principle of antisense RNA to eliminate plasmids is: we place antisense RNA that can specifically bind to the mRNA of key metabolic genes such as TCA cycle-related genes, protein synthesis-related genes, DNA / RNA synthesis-related genes, amino acid synthesis-related genes, ribose synthesis-related genes, ribosome synthesis-related genes, and ribosome assembly-related genes downstream of the inducible promoter, and control the transcription of antisense RNA of key metabolic genes by adding / not adding inducers. When the antisense RNA is transcribed, the antisense RNA can bind to the mRNA of the key metabolic gene in the form of hydrogen bond pairing, thereby inhibiting the translation of the key metabolic gene, so that the growth rate of cells expressing antisense RNA is significantly lower than that of cells that do not express antisense RNA. Due to the spontaneous loss of plasmids, plasmid elimination can be achieved by enriching plasmid-free cells through subculture. (Principle as Figure 4 (shown)

[0110] The cascade reaction neutralizes the antisense RNA through the antisense neutralizing RNA to eliminate the inhibition of cell growth by the antisense RNA expressed by the plasmid to be eliminated, thus achieving controllable plasmid-free cell enrichment and plasmid elimination.

[0111] Instruments and equipment:

[0112] 1.PCR instrument

[0113] 2. Electrophoresis apparatus

[0114] 3. Electrophoresis tank

[0115] 4. Ultra-low temperature refrigerator

[0116] 5. Biochemical incubator

[0117] 6. Autoclave

[0118] 7. Water bath

[0119] 8. Medical freezer

[0120] 9. Desktop Centrifuge

[0121] 10. Pipette

[0122] 11. Oven

[0123] 12.Electroshock converter and its matching 1mm / 2mm electroshock cup

[0124] Reagents:

[0125]

[0126]

[0127] Biomaterials:

[0128] E. coli related plasmids and primers:

[0129] Primers:

[0130] EP001:CAGCATCCTGCGATGCAGATCCGGAAC (SEQ ID NO: 23)

[0131] EP002:CAATCCGGATATAGTTCCTCCTTTCAGC (SEQ ID NO: 24)

[0132] EP003: GGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGC (SEQ ID NO: 25) EP004: CTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTAC (SEQ ID NO: 26) EP005: GGAAGGGAAGAAAGCGAAAGGAGC (SEQ ID NO: 27)

[0133] EP006:GGAACGTTGTGAGGGTAAACAACTGGC (SEQ ID NO: 28)

[0134] Gene sequences commissioned for synthesis:

[0135] CAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGAGAGCGTTCACCGACAAA

[0136] CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCGATGA

[0137] AGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGCGAAGCCGCTAGCA (omitted here, see the sequence list for details)

[0138] CATCCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTA

[0139] AAGGCGAAGCCAAATCCCCCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGG GTTTTTGCTGAAAGGAGGAACTATATCCGGATTG (SEQ ID NO: 29)

[0140] Corynebacterium glutamicum related plasmids and primers:

[0141] Primers:

[0142] GP001: CCATGAACAGAAATCCCGTAAATGCATGCCGCTTCGCCTTCG (SEQ ID NO: 30) GP002: GAGCAGTATTTTGCGCGCCCACCGCAGTAGGCGCAGCTGATGCGATC (SEQ ID NO: 31)

[0143] GP003:GGATTTCTGTTCATGGGGGTAATG (SEQ ID NO: 32)

[0144] GP004:GCGCGCAAAATACTGCTCAGACG (SEQ ID NO: 33)

[0145] GP005:GCTTACAGACAAGCTGTGACCGTCTC (SEQ ID NO: 34)

[0146] GP006:TTGTGGCCGACCTTGAAGAGCTGG (SEQ ID NO: 35)

[0147] The sequence of the commissioned synthesis:

[0148] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACC

[0149] TGCATCGATTTATTAGGACAGCTTCACTGCCACATCGTTCACCTTTTCTTCCGCAGCCTGC

[0150] GCGGAATTCGGATGGGGATGCGCCGGTGCACTTCTTGAACACGCGGGAGAAGTACAGC (omitted here, see the sequence list for details)

[0151] TCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTA

[0152] ATTCCGCTAGATGACGTGCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGCGCTCG CGGCTGGCTGCGGCACGACACGCGTCTGAGCAGTATTTTGCGCGC (SEQ ID NO: 36) Saccharomyces cerevisiae related plasmids and primers:

[0153] Primers:

[0154] SP001: TGGCGAGAAAGGAAGGGAAGAAAGC (SEQ ID NO: 37)

[0155] SP002:TGGCCGATTCATTAATGCAGGGCC (SEQ ID NO: 38)

[0156] SP003:CCCTTCCTTTCTCCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT (SEQ ID NO: 39) SP004: GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGG (SEQ ID NO: 40)

[0157] SP005: CCGTAAAGCACTAAATCGGAACCC (SEQ ID NO: 41)

[0158] SP006:CTCACATGTTCTTTCCTGCGTTATCC (SEQ ID NO: 42)

[0159] Delegated Synthesis Sequence

[0160] TGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGA

[0161] GCTTGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAA

[0162] TGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAAC (omitted here, see the sequence list for details)

[0163] TTATTTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTT

[0164] CTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTG

[0165] GGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCA (SEQ ID NO: 43)

[0166] Example 1 Construction of Escherichia coli Arabinose Promoter Plasmid Elimination Module

[0167] (1) High-fidelity enzyme amplification and plasmid construction experiments:

[0168] 1. Primers EP001 and EP002 were used to amplify the synthetic sequence SEQ ID NO: 29 according to the following system and PCR procedure, and primers EP003 and EP004 were used to amplify the plasmid template pet28a plasmid according to the same system and PCR procedure.

[0169] system:

[0170]

[0171] PCR procedure:

[0172]

[0173] 2. Use the ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products EP001-EP002 and EP003-EP004 according to the instructions of the purification kit.

[0174] 3. Connect the two fragments of the purified product EP001-EP002 / EP003-EP004 according to the operation flow in the instruction manual of the ready-to-use seamless cloning kit.

[0175] 4. Follow the steps below to convert 5αChemicallyCompetentCell after overnight culture:

[0176] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (gently pipette or tap the tube wall several times), and let it stand on ice for 5 minutes.

[0177] 2) Heat shock in a 42°C water bath for 45-60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes.

[0178] 3) Add 700 μL of sterile liquid culture medium (SOB or LB) without antibiotics into the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 40 min.

[0179] 4) Take an appropriate volume of the resuscitation solution and spread it evenly on the LB solid medium containing 50ug / mL kanamycin. Incubate the medium at 37℃.

[0180] Incubate overnight.

[0181] (2) Construction result sequencing verification experiment:

[0182] The single colony obtained from the coated plate was sent for sequencing using primers EP005 and EP006 to verify the construction results.

[0183] Plasmid construction results:

[0184] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT (omitted here, see the sequence list for details)

[0185] CTCTACTGTTTCTCCATCCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCT

[0186] GGCTGCTATCGCTAAAGGCGAAGCCAAATCCCCCTAGCATAACCCCTTGGGGCCTCTAA ACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ ID NO: 44)

[0187] The antisense RNA sequence:

[0188] GGCTTCGCCTTTAGCGATAGCAGCCAGGAAGCCTGCTTTGACATAGGCGGCTTC G(SEQ IDNO: 45)

[0189] Antisense RNA neutralizing sequence:

[0190] CCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGCGAAGCCAAATCCCC (SEQ ID NO: 46)

[0191] (3) Plasmid extraction experiment:

[0192] According to the sequencing results in (2), a single colony available on the transformation plate in (1) was selected to inoculate 5 mL of LB liquid culture medium containing 50 ug / mL kanamycin for overnight activation, and plasmid extraction was completed according to the instructions of the Tiangen Plasmid Extraction Kit.

[0193] Example 2 Construction of the Corynebacterium glutamicum Arabinose Promoter Plasmid Elimination Module

[0194] (1) High-fidelity enzyme amplification and plasmid construction experiments:

[0195] 1. Primers GP001 and GP002 were designed to amplify the synthesized fragment (SEQ ID NO: 36) according to the following system and PCR procedure; primers GP003 and GP004 were designed to amplify the pEC-XK99E plasmid according to the following system and PCR procedure.

[0196] system:

[0197]

[0198] PCR procedure:

[0199]

[0200] 2. Use the ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products GP001-GP002 / GP003-GP004 according to the instructions of the purification kit.

[0201] 3. Connect the purified products GP001-GP002 / GP003-GP004 according to the operation procedures in the instructions of the ready-to-use seamless cloning kit.

[0202] 4. Follow the steps below to convert 5αChemicallyCompetentCell after overnight culture:

[0203] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (gently pipette or tap the tube wall several times), and let it stand on ice for 5 minutes.

[0204] 2) Heat shock in a 42°C water bath for 45-60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes.

[0205] 3) Add 700 μL of sterile liquid culture medium (SOB or LB) without antibiotics into the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 40 min.

[0206] 4) Take an appropriate volume of the resuscitation solution and evenly spread it on LB solid culture medium containing 50ug / mL kanamycin, and culture it upside down in a 37°C incubator overnight.

[0207] (2) Construction result sequencing verification experiment:

[0208] Take a single colony obtained from the coated plate and send it for sequencing with GP005 / GP006 primers to verify the construction results.

[0209] Plasmid construction results:

[0210] CCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATCGTGGAGGCTTTCGGCCGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGCGCAACGAGGAGCGTTACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCCCAAATAAAACGAAAGGCTCAGT (omitted here, see the sequence list for details)

[0211] AGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGC

[0212] TATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT (SEQ ID NO: 47)

[0213] The antisense RNA sequence:

[0214] CCCAACGCAGGAACTGGTAACGCTCCTCGTTGCGCTCGTACTCGATCTCAACGTTC TTAGCCAGTGCATCTGGGCGGCCGAAAGCCTCCACGATGACGGAGTGGTCAATGACCAT CTCGG (SEQ ID NO: 48)

[0215] Antisense RNA neutralizing sequence:

[0216] CCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATCGTGGAGGCTTTCG GCCGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGCGCAACGAGGAGCG TTACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCC (SEQ ID NO: 49)

[0217] (3) Plasmid extraction experiment:

[0218] According to the sequencing results in (2), a single colony available on the transformation plate in (1) was selected to inoculate 5 mL of LB liquid culture medium containing 50 ug / mL kanamycin for overnight activation, and plasmid extraction was completed according to the instructions of the Tiangen Plasmid Extraction Kit.

[0219] Example 3 Construction of Saccharomyces cerevisiae inducible GAL1 promoter plasmid elimination module

[0220] (1) High-fidelity enzyme amplification and plasmid construction experiments:

[0221] 1. Design primers SP001-SP002 to amplify the synthesized fragment SEQ ID NO: 43 according to the following system and PCR program; primers SP003-SP004 to amplify the yeast 2um plasmid.

[0222] system:

[0223]

[0224] PCR procedure:

[0225]

[0226] 2. Use the ready-to-use seamless cloning kit to assemble the SanPrep column-based PCR product purification kit, and purify the PCR products SP001-SP002 / SP003-SP004 according to the instructions of the purification kit.

[0227] 3. Connect the purified products SP001-SP002 / SP003-SP004 respectively according to the operation procedures in the instructions of the ready-to-use seamless cloning kit.

[0228] 4. Follow the steps below to convert 5αChemicallyCompetentCell after overnight culture:

[0229] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (gently pipette or tap the tube wall several times), and let it stand on ice for 5 minutes.

[0230] 2) Heat shock in a 42°C water bath for 45-60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes.

[0231] 3) Add 700 μL of sterile liquid culture medium (SOB or LB) without antibiotics into the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 40 min.

[0232] 4) Take an appropriate volume of the resuscitation solution and evenly spread it on the LB solid culture medium containing 100ug / mL AMP, and culture it upside down in a 37℃ incubator overnight.

[0233] (2) Construction result sequencing verification experiment:

[0234] Single colonies obtained from two coated plates were sent for sequencing with primers SP003 / SP004 to verify the construction results and obtain the correct plasmid for sequencing.

[0235] Plasmid construction results:

[0236] CAACTTTGTATAGAAAAGTTGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGT (omitted here, see the sequence list for details)

[0237] GGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGAAACA

[0238] Antisense RNA sequence involved in TTTTGAAGCTATGGTGTGTGGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCG CGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT (SEQ ID NO: 50):

[0239] GGAGCCTTCAATTCCCATGGACGACCTGCCATAACATCAACGGCATCAGCACCACCAACACCAATAGCCAATTGAC (SEQ ID NO: 51)

[0240] Antisense RNA neutralizing sequence:

[0241] GGTGGTTTAGGTCAATTGGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATG GCAGGTCGTCCATGGGAATTGAAGGCTCCAAAGATCTTAGG. (SEQ ID NO: 52)

[0242] (3) Plasmid extraction experiment:

[0243] According to the sequencing results in (2), a single colony available on the transformation plate in (1) was selected and inoculated into 5 mL of LB liquid culture medium containing 100 ug / mL AMP for overnight activation, and plasmid extraction was completed according to the instructions of the Tiangen Plasmid Extraction Kit.

[0244] Example 4 Plasmid elimination using the E. coli cascade reaction plasmid elimination module

[0245] 1. Make the E. coli to be transformed competent according to the process described in the super competent cell preparation kit.

[0246] 2. Take 100 μL of competent cells melted on ice, add the plasmid described in the technical solution "Construction experiment of Escherichia coli cascade reaction plasmid elimination module", gently mix (gently blow or tap the tube wall several times), and let it stand on ice for 5 minutes.

[0247] 3. Heat shock in a 42°C water bath for 60 seconds, quickly transfer to an ice bath, and let stand for 2 minutes (do not shake the sample during standing on ice, otherwise it will reduce the transformation efficiency).

[0248] 4. Add 700 μL of sterile liquid culture medium (SOB or LB) without antibiotics but containing 1% arabinose to the centrifuge tube and mix well.

[0249] 5. Take an appropriate volume of the recovery solution and spread it evenly on the LB solid culture medium containing 50ug / mL kanamycin and 1% arabinose, and culture it upside down in a 37℃ incubator overnight.

[0250] 6. Add the E. coli constructed in "5" or the frozen E. coli containing the building blocks in "5" into LB liquid medium containing no more than 1 / 5 of the volume of the conical flask, at 37°C, 220rpm. After overnight culture (12h), streak on an antibiotic-free LB solid medium plate.

[0251] 7. Take the single colony obtained in "6" and spot it on the solid culture medium plates with and without antibiotics (a single colony needs to be spotted twice to ensure the phenotypic difference).

[0252] Specific results such as Figure 1As shown in the figure, after plasmid elimination, most of the bacteria have lost their resistance to antibiotics and can no longer grow on antibiotic plates, indicating that the relevant resistance genes have been lost and the plasmid has been successfully eliminated. The corresponding strain with successful elimination can be inoculated to obtain the strain with successful plasmid elimination.

[0253] Example 5

[0254] 1. Activate the Corynebacterium glutamicum ATCC13032 strain overnight with 5 ml of brain heart infusion liquid culture medium.

[0255] 2. Inoculate all activated Corynebacterium glutamicum bacterial liquid into 45 mL Epo liquid culture medium until the final OD600 reaches 0.2, and grow at 200 rpm and 30°C until OD600 reaches 0.9.

[0256] 3. Transfer the bacterial solution to a sterile 50 mL centrifuge tube and centrifuge to remove the supernatant. Wash 4 times with ice-cold 10% sterile glycerol. Dispense 100 ul / tube into sterile 1.5 mL centrifuge tubes and store at 70°C or use immediately.

[0257] 4. During electroporation, the competent cells were aliquoted and thawed on ice, and 5uL of the plasmid obtained in the "Construction of Corynebacterium glutamicum Cascade Reaction Plasmid Elimination Module Experiment" was added.

[0258] 5. Transfer the mixture to a cold electroporation cup (0.1 cm) and perform an electroporation at 1.8 kV and 5 ms pulses. Immediately after electroporation, add 1 mL of brain heart infusion medium containing 1% arabinose to the cuvette, mix well, and transfer to a 1.5 mL centrifuge tube.

[0259] 6. Incubate at 30°C for 1 hour.

[0260] 7. Finally, spread the plate on a brain heart infusion agar plate containing 50ug / mL kanamycin and 1% arabinose.

[0261] 8. Add the frozen bacteria / puncture bacteria of Corynebacterium glutamicum constructed in "7" or Corynebacterium glutamicum containing the building blocks in "7" to a liquid culture medium containing antibiotic-free heart infusion solution whose volume of culture medium does not exceed 1 / 5 of the volume of the conical flask, and culture overnight (18 h) at 30°C and 220 rpm.

[0262] 9. Streak on an antibiotic-free LB solid medium plate.

[0263] 10. Take the single colony obtained in "9" and spot it on the solid culture medium plates with and without antibiotics (a single colony needs to be spotted twice to ensure the phenotypic difference).

[0264] Specific results such as Figure 2As shown, after plasmid elimination, most of the bacteria lost their resistance to antibiotics and could no longer grow on antibiotic plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully eliminated. By inoculating the corresponding successfully eliminated strain, the strain with successfully eliminated plasmids could be obtained.

[0265] Example 6 Plasmid elimination using the Saccharomyces cerevisiae cascade reaction plasmid elimination module

[0266] Prepare the following reagents first:

[0267] 10*TE buffer

[0268] 100mM LiAC buffer

[0269] 1M DTT buffer

[0270] ddH2O

[0271] 1M Sorbitol Solution

[0272] Preparation of competent culture of Saccharomyces cerevisiae:

[0273] 1. Precool reagents.

[0274] 2. Take 30 μL of the Saccharomyces cerevisiae glycerol tube and inoculate it into 10 mL of liquid YPD medium. Culture it in a constant temperature shaker at 30°C for 24 h. Then take 100 μL of it and inoculate it into 100 mL of liquid YPD medium until the bacterial concentration OD600 grows to 1.3-1.5.

[0275] 3. Divide the bacterial solution into three pre-cooled 50 mL centrifuge tubes, centrifuge at 4°C, 5000 r×min-1 for 5 min, discard the supernatant and collect the bacteria.

[0276] 4. Use 4 mL of pre-cooled ddH2O to blow and aspirate the bacteria to resuspend them. Combine the three tubes into one tube and add 2 mL of pre-cooled 10×TE buffer, 2 mL of 10×LiAc buffer and 0.5 mL of 1M DTT buffer. Mix by pipetting and aspirate. Place the centrifuge tube in a constant temperature water bath shaker at 30°C and 50 r×min-1 and culture for 45 min.

[0277] 5. Add 13.5 mL of pre-cooled ddH2O into a 50 mL centrifuge tube, centrifuge at 4°C, 5000 r×min-1 for 5 min, discard the supernatant and collect the bacteria.

[0278] 6. Add 25 mL of pre-cooled ddH2O into a 50 mL centrifuge tube, blow and aspirate the suspended bacteria, centrifuge at 5000 r×min-1 for 5 min, discard the supernatant, collect the bacteria, and then wash the bacteria twice with 1 mol·L-1 sorbitol (25 mL of sorbitol for the first time and 20 mL for the second time).

[0279] 7. Add 1 mL of pre-cooled sorbitol to the centrifuge tube to suspend the bacteria, and dispense into 1.5 mL EP tubes, 80 μL per tube, for immediate use.

[0280] 8. During electroporation, place the aliquoted competent cells on ice and add 5uL of the plasmid obtained in the "Saccharomyces cerevisiae cascade reaction plasmid elimination module construction experiment".

[0281] 9. Transfer the mixture to a cold electroporation cup (0.2 cm) and perform an electroporation at 1.5 kV, 200 Ω, 25 uF, and 5 ms pulses. Immediately after electroporation, add 1 mL of YPD medium containing 2% galactose without antibiotics to the cuvette, mix well, and transfer to a 1.5 mL centrifuge tube. Incubate at 30°C and 200 rpm on a shaker for 1 to 3 hours.

[0282] 10. Collect the bacteria by centrifugation. Spread 150ul of the bacterial solution on a 500ug / mL neomycin sulfate resistance plate containing 2% galactose and culture at 30℃ for 2-3 days.

[0283] 11. Add the Saccharomyces cerevisiae constructed in "10" or the frozen / punctured Saccharomyces cerevisiae containing the building blocks in "10" into an antibiotic-free YPD liquid culture medium containing a culture medium volume not exceeding 1 / 5 of the volume of the conical flask, culture at 30°C, 220 rpm for 48 h, and then streak on an antibiotic-free YPD solid culture medium plate.

[0284] Take the single colony obtained in "11" and spot it on the YPD solid culture medium plates with and without antibiotics (neomycin sulfate 500ug / mL) respectively (a single colony needs to be spotted twice to ensure the phenotypic difference can be distinguished).

[0285] Specific results such as Figure 3 As shown, after plasmid elimination, most of the bacteria lost their resistance to antibiotics and could no longer grow on antibiotic plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully eliminated. By inoculating the corresponding successfully eliminated strain, the strain with successfully eliminated plasmids could be obtained.

[0286] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An application of a plasmid elimination module based on cascade reaction, characterized in that: Used for the selective elimination of a designated target plasmid or the selective simultaneous elimination of several designated target plasmids.

2. The use according to claim 1, characterized in that: The plasmid elimination module includes three types of independent expression regions, one of which is an antisense RNA expression region, which includes antisense RNA, a promoter and a terminator of the antisense RNA; the antisense RNA is the antisense RNA of the metabolic key gene mRNA, which can bind to the metabolic key gene mRNA to hinder the translation of the metabolic key gene mRNA; one type of region is an antisense neutralizing RNA expression region, which includes antisense neutralizing RNA, an inducible promoter and a terminator of the antisense neutralizing RNA; the last type of region is a transcription factor region, which is composed of a constitutive promoter of the transcription factor fragment, which includes an RNA polymerase binding site and a transcription start site, a ribosome binding site, a transcription factor translation region, and a terminator; the antisense neutralizing RNA is complementary to the antisense RNA sequence, can bind to the antisense RNA so that it cannot bind to the metabolic key gene mRNA and inhibit the antisense RNA from producing an RNA fragment that hinders the translation of the metabolic key gene mRNA.

3. The use according to claim 2, characterized in that: The transcription factor is a protein that can induce transcription of downstream genes of the binding site after binding to a specific compound; or a protein that can induce transcription of downstream genes before binding to a specific compound but cannot induce transcription of downstream genes of the binding site after binding to the specific compound.

4. The use according to claim 3, characterized in that: The transcription factor is an arabinose transcription factor.

5. The use according to claim 2, characterized in that: The constitutive promoter of the transcription factor fragment is the constitutive promoter of the arabinose transcription factor.

6. The use according to claim 2, characterized in that: The antisense RNA promoter includes J23119 promoter, T7, tet, trc, trp, tac and constitutive promoters related to chloramphenicol, kanamycin and penicillin resistance genes commonly used in glutamicum antibiotics, or TEF1 promoter, and the antisense RNA terminator includes Cyc1 terminator, rrnB T1 terminator or rrnB T2 terminator.

7. The plasmid elimination module according to claim 2, characterized in that: The antisense neutralizing RNA inducible promoter includes GAL1 promoter and arabinose promoter, and the antisense RNA terminator includes Cyc1 terminator, rrnB T1 terminator or T7 terminator.

8. The use according to claim 2, characterized in that: The plasmid elimination module is mounted in the plasmid to be eliminated.

9. The use according to claim 2, characterized in that The plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and yeast 2μ plasmid.

10. The use according to claim 2, characterized in that: The key metabolic genes are TCA cycle related genes, protein synthesis related genes, aerobic respiration related genes, anaerobic respiration related genes, lipid synthesis related genes, cell wall synthesis related genes, sugar transport pathway related genes, amino acid transport pathway related genes, ATP synthesis related genes, cytoskeleton synthesis related genes, NADPH / NADP + Synthesis-related genes, NADH / NAD + Synthesis-related genes, vitamin synthesis-related genes, one-carbon transport system-related genes, carbon dioxide fixation-related genes, DNA / RNA synthesis-related genes, amino acid synthesis-related genes, ribose synthesis-related genes, ribosome synthesis-related genes, ribosome assembly-related genes, aminoacyl-tRNA synthetase-related genes and isoenzyme genes of the above genes.

11. The use according to any one of claims 1 to 10, characterized in that: The objects of elimination of the plasmid elimination module are all microorganisms, animals or plant cells that have stable free plasmids, free plasmids that have a certain probability of not being distributed to daughter cells, and free plasmids that will not cause cell death or proliferation failure after elimination.

Images