Plasmid elimination module based on cascade reaction

Abstract

The invention relates to the field of gene engineering, in particular to a cascade reaction-based plasmid elimination module, which comprises three types of mutually independent expression regions, one type of the expression regions is an antisense RNA expression region and comprises antisense RNA, a promoter of the antisense RNA and a terminator, and the other type of the expression regions is an antisense RNA expression region and comprises antisense RNA, a promoter of the antisense RNA and a terminator of the antisense RNA. The antisense RNA is the antisense RNA of a metabolic key gene mRNA, and can be combined with the metabolic key gene mRNA to hinder the translation of the metabolic key gene mRNA; the first region is an antisense neutralizing RNA expression region and comprises antisense neutralizing RNA, an inducible promoter of the antisense neutralizing RNA and a terminator of the antisense neutralizing RNA; the last type of region is a transcription factor region and is composed of constitutive promoters carried by a transcription factor fragment, and the constitutive promoters comprise an RNA polymerase binding site, a transcription start site, a ribosome binding site, a transcription factor translation region and a terminator; the antisense neutralizing RNA is an RNA fragment which is complementary with the antisense RNA in sequence, can be combined with the antisense RNA to enable the antisense RNA not to be combined with the metabolic key gene mRNA, and inhibits the antisense RNA from generating translation obstruction on the metabolic key gene mRNA. 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 present invention relates to the field of genetic engineering, and particularly to a plasmid elimination module based on a cascade reaction. Background Art

[0002] Episomal plasmids are a common 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, after using a genome editing or gene circuit plasmid to achieve the purpose of genome editing or gene circuit design, the genome editing tool or gene circuit carried on the plasmid becomes an unstable factor in the cell genome. At this time, it is necessary to eliminate the plasmid to obtain plasmid-free cells. The current plasmid elimination techniques are mainly divided into three types, namely temperature-sensitive replicon plasmid elimination, sacB plasmid elimination, and CRISPR protein-related plasmid elimination techniques.

[0003] The elimination efficiency of temperature-sensitive replicon plasmids is relatively low and is commonly used for low-copy plasmid elimination, but it has poor effects on medium- and high-copy plasmid elimination. Moreover, generally, the same replicon in the same cell can only serve one plasmid at the same time. If a temperature-sensitive replicon is used to serve multiple plasmids, it may exacerbate the spontaneous loss of plasmids, 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 simultaneously. However, when multiple plasmids are present in the cell, the plasmid elimination method based on the sacB gene cannot accurately eliminate specific types of plasmids, and its tolerance to mutations is weak, making it prone to failure;

[0005] CRISPR protein-related plasmid elimination. The plasmid elimination technique based on CRISPR proteins can eliminate multiple plasmids simultaneously and freely select the types of plasmids to be eliminated. However, the plasmid elimination technique based on CRISPR proteins has the risks of CRISPR protein gene mutation failure or CRISPR protein cleavage off-target, and the gene sequence of CRISPR-related protein elements is relatively 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 cell, it is necessary to additionally transfer a plasmid carrying the CRISPR protein, and the self-targeting elimination failure rate of the plasmid carrying the CRISPR protein is relatively high. Summary of the Invention

[0006] The present invention proposes a plasmid elimination technology based on antisense RNA, designs 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 smaller 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] According to an embodiment of the present invention, a plasmid elimination module based on cascade reaction includes three types of independent expression regions, one of which is an antisense RNA expression region, including 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, including 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 an RNA fragment that hinders the translation of the key metabolic gene mRNA.

[0012] According to an embodiment of the present invention, the transcription factor described is a protein that can induce the transcription of genes downstream of the binding site after binding to a specific compound; or a protein that can induce the transcription of downstream genes before binding to a specific compound and cannot induce the transcription of genes downstream of the binding site after binding to the specific compound.

[0013] According to an embodiment of the present invention, the transcription factor described is an arabinose transcription factor.

[0014] According to an embodiment of the present invention, the constitutive promoter carried by the transcription factor fragment is the constitutive promoter carried by the arabinose transcription factor.

[0015] According to an embodiment of the present invention, the antisense RNA promoter includes the J23119 promoter, T7, tet, trc, trp, tac, and constitutive promoters related to the common antibiotics chloramphenicol, kanamycin, and penicillin resistance genes of Bacillus glutamicum, or the TEF1 promoter, and the antisense RNA terminator includes the Cyc1 terminator, rrnB T1 terminator, or rrnB T2 terminator.

[0016] According to an embodiment of the present invention, the inducible promoter of the antisense neutralizing RNA includes the GAL1 promoter and the arabinose promoter, and the antisense RNA terminator includes the Cyc1 terminator, rrnB T1 terminator, or T7 terminator.

[0017] According to an embodiment of the present invention, the plasmid elimination module is carried on the plasmid to be eliminated.

[0018] According to an embodiment of the present invention, the plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and yeast 2μ plasmid.

[0019] According to an embodiment of the present invention, the key metabolic genes are genes related to the TCA cycle, protein synthesis, aerobic respiration, anaerobic respiration, lipid synthesis, cell wall synthesis, sugar transport pathway, amino acid transport pathway, ATP synthesis, cytoskeleton synthesis, 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 isozyme genes of the above genes.

[0020] According to the embodiments of the present invention, the elimination target of the plasmid elimination module is all microorganisms, animal or plant cells that have stable free plasmids, where there is a certain probability that the free plasmids will not be distributed to daughter cells, and the elimination of the free plasmids will not cause cell death or inability to proliferate.

[0021] The design concept of the plasmid elimination module proposed by the present invention is the core of the invention's creativity. This design realizes plasmid elimination by inducing an increase in the growth rate difference between plasmid-containing and plasmid-free cells. It can be applied to all plasmids with spontaneous loss but low spontaneous loss efficiency. The characteristics of the cascade reaction make it convenient to form a multiple signal system for sequential biological behavior control.

[0022] To facilitate the understanding of the present invention by those skilled in the art, the main vocabulary explanations are as follows:

[0023] Gene circuit: A combination of DNA sequences that arranges specific gene elements such as promoters, terminators, protein translation regions, and specific protein binding regions in a certain pattern at single or multiple sites to achieve specific functions.

[0024] Convenient to use: This module can be stored in the form of PCR products / plasmids, etc., and can be inserted into plasmids outside or inside cells through forms such as Gibson assembly, Golden Gate cloning, enzymatic digestion and ligation, and genome editing.

[0025] Programmable: The use of this module is not restricted by the plasmid copy number, and selective elimination can be achieved in cells containing multiple plasmids by combining different regulatory proteins.

[0026] Wide adaptability: This module can be used across species by optimizing codons / replacing related control elements.

[0027] 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.

[0028] Short required sequence: When using the cell's native regulatory factors, the total length of the plasmid elimination module sequence proposed by the present invention is usually less than 1 kbp, and even less than 100 bp. When using its own regulatory factors, the total length of the plasmid elimination module sequence proposed by the present invention usually does not exceed 3 kbp.

[0029] Constructed plasmid: It mainly refers to a tool plasmid that constructs the essential functional proteins for genome editing to assist in achieving the purpose of genome editing of genetically engineered cells. Such plasmids usually need to be eliminated after completing the genome editing of engineered cells to avoid introducing or causing additional mutations in the application of engineered cells.

[0030] Artificially constructed plasmid: It mainly refers to a plasmid that does not involve genome editing and is artificially constructed to express homologous / heterologous proteins or some functional gene circuits.

[0031] Antisense neutralizing RNA: Complementary to the antisense RNA sequence, it can bind to the antisense RNA, preventing it from binding to the mRNA of key metabolic genes and inhibiting the hindrance of the antisense RNA to the translation of the mRNA of key metabolic genes.

[0032] Temporality: With a controllable time difference.

[0033] Free plasmid: A linear or circular double-stranded DNA that can autonomously assist in replication in cells or co-replicate with the genome, can be distributed to daughter cells along with cell proliferation, and can stably exist outside the genome.

[0034] Plasmid backbone: The DNA sequence carrying the plasmid replicon and plasmid maintenance genes.

[0035] Constitutive promoter: A specific DNA sequence that can be recognized by the Escherichia coli σ factor and recruit RNA polymerase subunits for transcription downstream.

[0036] An example is as follows:

[0037] Escherichia coli-related sequences:

[0038] Full sequence of the interference region of the Escherichia coli acnB gene:

[0039]

[0040] Full sequence of pET28a plasmid:

[0041] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTA

[0042] CGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC

[0043] CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT

[0044] TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG

[0045] GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA

[0046] CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCT

[0047] ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT

[0048] TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCAC

[0049] TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

[0050] TATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA

[0051] TTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA

[0052] ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC

[0053] GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA

[0054] AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC

[0055] AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAA

[0056] CCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

[0057] CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAA

[0058] TATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGC

[0059] AGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG

[0060] GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT

[0061] ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGAT

[0062] TGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC

[0063] ATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA

[0064] CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAA

[0065] CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0066] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

[0067] CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC

[0068] AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

[0069] AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

[0070] GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

[0071] GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

[0072] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

[0073] AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0074] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

[0075] CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA

[0076] CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

[0077] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

[0078] ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0079] GAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATG

[0080] GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTAT

[0081] CGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC

[0082] TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG

[0083] CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA

[0084] GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCT

[0085] CGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGG

[0086] CGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG

[0087] TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT

[0088] GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0089] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCA

[0090] CAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA

[0091] CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTC

[0092] AGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCAT

[0093] TCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCAC

[0094] GATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAAC

[0095] GTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATAC

[0096] CGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATG

[0097] ACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAG

[0098] TGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT

[0099] CTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCG

[0100] TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0101] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA

[0102] CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG

[0103] CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACG

[0104] GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACC

[0105] AACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG

[0106] CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0107] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTG

[0108] AGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC

[0109] TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTAC

[0110] CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAAT

[0111] AACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0112] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC

[0113] AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0114] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG

[0115] TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA

[0116] ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG

[0117] CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGAC

[0118] ATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT

[0119] CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0120] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG

[0121] CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGG

[0122] GCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGA

[0123] TCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGT

[0124] GATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAAT

[0125] ACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT

[0126] TAACTTTAAGAAGGAGATATACCATGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC

[0127] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:2)

[0128] Arabinose promoter sequence (the arrow indicates the transcription start direction):

[0129] AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ IDNO:3)

[0130] T7 terminator sequence (the arrow indicates the transcription termination direction):

[0131] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO:4)

[0132] Arabinose transcription factor expression cassette (including promoter, CDS region, terminator):

[0133]

[0134] J23119 promoter:

[0135] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC → (SEQ ID NO: 6)

[0136] rrnB T1 terminator:

[0137] CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC (SEQ ID NO: 7)

[0138] Corynebacterium glutamicum - related sequence:

[0139] Full - length CDS region sequence of the acn gene of Corynebacterium glutamicum:

[0140]

[0141] Glutamic acid corynebacterium pEC-XK99E plasmid sequence:

[0142] GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCC

[0143] CGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTT

[0144] GCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG

[0145] AATTAATTCCGCTAGATGACGTGCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGC

[0146] GCTCGCGGCTGGCTGCGGCACGACACGCGTCTGAGCAGTATTTTGCGCGCCGTCCTCGT

[0147] GGGTCAGGCCGGGGTGGGATCAGGCCACCGCAGTAGGCGCAGCTGATGCGATCCTCCA

[0148] CTACTGCGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCGGCCAGCTCTTCAAGGT

[0149] CGGCCACAAGCGTTTCTAGGTCGCTCGCGGCACTTGCCCAGTCGCGTGATGCTGGCGCG

[0150] TCTGTCGTATCGAGGGCGCGGAAAAATCCGATCACCGTTTTTAAATCGACGGCGGCATC

[0151] GAGTGCGTCGGACTCCAGCGCGACATCGGAGAGATCCACCGCTGATGCTTCAGGCCAG

[0152] TTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTTAAAAATTCT

[0153] AGCCCCAATTCTGATAATTTCTTCCGGCACTCCTGCGAAAACCTGCGAGACTTCTTGCCC

[0154] AGAAAAAACGCCAAGCGCAGCGGTTACCGCACTTTTTTTCCAGGTGATTTCACCCTGAC

[0155] CAGCGAAGCGGCACTTTAGTGCATGAGGTGTGCCCCTGGTTTCCCCTCTTTGGAGGGTT

[0156] CAACCCAAAAAAGCACACAAGCAAAAATGAAAATCATCATGAGCAAGTTGGTGCGAAG

[0157] CAGCAACGCGCTAGCTCCAAAAAGGTCTCCAGGATCTCGAGGAGATTTTTGAGGGGGA

[0158] GGGAGTCGAGGAAGAGCCAGAGCAGAAGGCGGGGAACCGTTCTCTGCCGACAGCGTG

[0159] AGCCCCCCTTAAAAATCAGGCCGGGGAGGAACCGGGGAGGGATCAGAGCTAGGAGCG

[0160] AGACACCCTAAAGGGGGGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAG

[0161] CCCGTGGATAGCGGAGGGTGAGGGCAAGTGAGAGCCAGAGCAAGGACGGGACCCCTA

[0162] AAGGGGGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGACG

[0163] GCCGCGTTTAGCTTCCATTCCAAGTGCCTTTCTGACTTGTTGGATGCGCCTTTCACTGAC

[0164] ACCTAGTTCGCCTGCAAGCTCACGAGTCGAGGGATCAGCAACCGATTGAGAACGGGCA

[0165] TCCAGGATCGCAGTTTTGACGCGAAGTTCGAGCAACTCGCCTGTCATTTCTCGGCGTTT

[0166] GTTTGCTTCCGCTAATCGCTGTCGCGTCTCCTGCGCATACTTACTTTCTGGGTCAGCCCAT

[0167] CTGCGTGCATTCGATGTAGCTGCGCCCCGTCGCCCCATCGTCGCTAGAGCTTTCCGCCCT

[0168] CGGCTGCTCTGCGTTTCCACCCGACGAGCAGGGACGACTGGCTGGCCTTTAGCCACGTA

[0169] GCCGCGCACACGACGCGCCATCGTCAGGCGATCACGCATGGCGGGAAGATCCGGCTCC

[0170] CGGCCGTCTGCACCGACCGCCTGGGCAACGTTGTACGCCACTTCATACGCGTCGATGAT

[0171] CTTGGCATCTTTTAGGCGCTCACCAGCAGCTTTGAGCTGGTATCCCACGGTCAACGCGT

[0172] GGCGAAACGCGGTCTCGTCGCGCGCTCGCTCTGGATTTGTCCAGAGCACTCGCACGCCG

[0173] TCGATCAGGTCGCCGGACGCGTCCAGGGCGCTCGGCAGGCTCGCGTCCAAAATCGCTA

[0174] GCGCCTTGGCTTCTGCGGTGGCGCGTTGTGCCGCTTCAATGCGGGCGCGTCCGCTGGAA

[0175] AAGTCCTGCTCAATGTACTTTTTCGGCTTCTGTGATCCGGTCATCGTTCGAGCAATCTCCA

[0176] TTAGGTCGGCCAGCCGATCCACACGATCATGCTGGCAGTGCCATTTATAGGCTGTCGGAT

[0177] CGTCTGAGACGTGCAGCGGCCACCGGCTCAGCCTATGCGAAAAAGCCTGGTCAGCGCC

[0178] GAAAACACGAGTCATTTCTTCCGTCGTTGCAGCCAGCAGGCGCATATTTGGGCTGGTTTT

[0179] ACCTGCTGCGGCATACACCGGGTCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAGCG

[0180] GATTCACGCCGATCCAAGCCGGCGCTTTTTCTAGGCGTGCCCATTTCTCTAAAATCGCGT

[0181] AGACCTGCGGGTTTACGTGCTCAATCTTCCCGCCGGCCTGGTGGCTGGGCACATCGATG

[0182] TCAAGCACGATCACCGCGGCATGTTGCGCGTGCGTCAGCGCAACGTACTGGCACCGCGT

[0183] CAGCGCTTTTGAGCCAGCCCGGTAGAGCTTTGGTTGGGTTTCGCCGGTATCCGGGTTTTT

[0184] AATCCAGGCGCTCGCGAAATCTCTTGTCTTGCTGCCCTGGAAGCTTTCGCGTCCCAGGT

[0185] GAGCGAGCAGTTCGCGGCGATCTTCTGCCGTCCAGCCGCGTGAGCCGCAGCGCATAGCT

[0186] TCGGGGTGGGTGTCGAACAGATCGGCGGACAATTTCCACGCGCTAGCTGTGACTGTGTC

[0187] CTGCGGATCGGCTAGAGTCATGTCTTGAGTGCTTTCTCCCAGCTGATGACTGGGGGTTAG

[0188] CCGACGCCCTGTGAGTTCCCGCTCACGGGGCGTTCAACTTTTTCAGGTATTTGTGCAGCT

[0189] TATCGTGTTTTCTTCGTAAATGAACGCTTAACTACCTTGTTAAACGTGGCAAATAGGCAG

[0190] GATTGATGGGGATCTAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAA

[0191] GGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGT

[0192] CAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCT

[0193] TGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACC

[0194] GGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGG

[0195] ATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGAC

[0196] AGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCG

[0197] CTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGAT

[0198] GCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCT

[0199] GTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACG

[0200] ACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGC

[0201] TGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAG

[0202] AAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGC

[0203] CCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCG

[0204] GTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTG

[0205] TTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGA

[0206] TGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGG

[0207] CCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTG

[0208] AAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCC

[0209] GATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGG

[0210] GGTTCGCGGAATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAG

[0211] ACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT

[0212] GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT

[0213] ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCC

[0214] TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACC

[0215] TCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG

[0216] GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGG

[0217] TTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC

[0218] GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT

[0219] AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG

[0220] GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC

[0221] TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT

[0222] GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT

[0223] AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG

[0224] CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

[0225] ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGC

[0226] ATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGA

[0227] CACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA

[0228] CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC

[0229] CGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTAC

[0230] GTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGA

[0231] GAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATG

[0232] CCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCG

[0233] AAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCG

[0234] TGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTG

[0235] GCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGG

[0236] TGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCG

[0237] GTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC

[0238] CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTC

[0239] TCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGC

[0240] GTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAG

[0241] TTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAAT

[0242] TCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACC

[0243] ATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGAT

[0244] GGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCT

[0245] CGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCACCA

[0246] TCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCT

[0247] CAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAA

[0248] CCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC

[0249] AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG

[0250] TGAGTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATG

[0251] CTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACT

[0252] GCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACAT

[0253] CATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATG(SEQ ID NO:9)

[0254] Arabinose promoter sequence (the arrow indicates the transcription start direction):

[0255] AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ IDNO:10)

[0256] T7 terminator sequence (the arrow indicates the transcription termination direction):

[0257] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO:11)

[0258] Arabinose transcription factor expression cassette (including promoter, CDS region, terminator):

[0259]

[0260] Trc promoter:

[0261] TTGACAATTAATCATCCGGCTCGTATAATG → (SEQ ID NO: 13)

[0262] rrnB T2 terminator:

[0263] → AGAAGGCCATCCTGACGGATGGCCTTTT (SEQ ID NO: 14)

[0264] Saccharomyces cerevisiae related sequence:

[0265] Full sequence of the CDS region of Saccharomyces cerevisiae ACO1 / GLU1 gene:

[0266] ATGCTGTCTGCACGTTCTGCCATCAAGAGACCCATTGTTCGTGGTCTTGCGACAGTCTCCAACTTGACTAGAGATTCAAAAGTCAACCAAAACTTATTAGAAGATCATTCTTTTATTAACTACAAGCAGAATGTGGAAACGCTGGATATCGTAAGAAAAAGATTAAACAGGCCATT

[0267] TACCTACGCGGAAAAGATTTTGTACGGTCACTTGGATGACCCTCATGGTCAAGATATTCA

[0268] GAGAGGTGTTTCATACCTAAAATTAAGACCAGATCGTGTTGCCTGTCAAGATGCTACTGC

[0269] TCAAATGGCTATTTTACAATTTATGTCCGCTGGTTTACCACAGGTTGCTAAGCCAGTCACT

[0270] GTCCACTGTGACCATTTGATTCAAGCACAAGTTGGTGGTGAAAAAGATTTGAAGAGAGC

[0271] TATAGATCTAAACAAGGAAGTTTATGATTTCTTGGCCTCTGCCACTGCGAAATATAACATG

[0272] GGTTTCTGGAAGCCAGGTTCCGGTATCATTCACCAAATTGTTCTGGAAAACTACGCTTTC

[0273] CCAGGTGCTTTGATCATTGGTACTGACTCCCATACACCAAATGCTGGTGGTTTAGGTCAA

[0274] TTGGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATGGCAGGTCGTCCATGGGAA

[0275] TTGAAGGCTCCAAAGATCTTAGGTGTTAAGTTGACTGGTAAGATGAACGGTTGGACTTC

[0276] TCCAAAGGATATTATTTTGAAATTGGCTGGTATCACAACTGTCAAAGGTGGTACTGGTAA

[0277] AATTGTTGAATATTTCGGTGATGGTGTTGACACCTTCTCCGCTACTGGTATGGGTACCATT

[0278] TGTAATATGGGTGCTGAAATCGGTGCTACCACATCTGTTTTCCCATTCAACAAATCTATGA

[0279] TTGAATATTTGGAAGCAACTGGTCGTGGTAAGATCGCTGACTTTGCTAAATTATACCACA

[0280] AGGACCTATTATCTGCTGATAAGGATGCTGAATACGATGAGGTCGTCGAAATTGACTTGA

[0281] ACACTCTGGAACCATACATCAATGGGCCATTTACCCCCGATTTGGCTACTCCAGTTTCTA

[0282] AGATGAAGGAAGTTGCTGTTGCTAATAACTGGCCATTGGATGTCAGAGTCGGTTTGATC

[0283] GGTTCTTGTACCAATTCCTCTTATGAAGATATGTCTCGTTCAGCATCCATTGTCAAGGATG

[0284] CTGCTGCTCATGGTTTGAAATCCAAGACCATTTTCACTGTTACTCCAGGTTCTGAACAAA

[0285] TCAGAGCCACTATTGAACGTGATGGCCAATTAGAAACCTTCAAAGAATTTGGTGGTATCG

[0286] TTTTGGCAAACGCCTGTGGCCCATGTATTGGTCAATGGGATCGTAGAGATATCAAGAAAG

[0287] GTGACAAGAATACTATCGTTTCCTCTTACAACAGAAATTTCACTTCTAGAAATGATGGTA

[0288] ACCCACAAACTCATGCTTTTGTTGCATCTCCAGAATTAGTAACTGCGTTCGCCATTGCGG

[0289] GTGATTTGAGATTCAACCCTCTAACAGACAAATTAAAGGACAAGGATGGTAATGAGTTC

[0290] ATGTTGAAACCACCACATGGTGATGGTTTGCCTCAAAGAGGTTATGATGCTGGTGAGAA

[0291] CACTTACCAAGCTCCACCTGCAGACCGTAGCACCGTTGAAGTTAAAGTTTCTCCAACTT

[0292] CAGACCGTCTACAACTGTTGAAACCATTCAAACCTTGGGATGGTAAGGATGCTAAAGAC

[0293] ATGCCAATCTTGATTAAGGCCGTCGGTAAGACAACTACTGATCATATTTCTATGGCTGGTC

[0294] CATGGTTGAAATACAGAGGTCATTTAGAAAACATTTCTAATAACTATATGATTGGTGCTAT

[0295] TAATGCTGAAAACAAGAAGGCTAACTGTGTTAAAAATGTATATACTGGTGAATACAAAG

[0296] GTGTTCCAGACACTGCTAGAGATTACAGAGACCAAGGTATCAAGTGGGTTGTTATTGGT

[0297] GATGAAAACTTTGGTGAAGGTTCCTCTCGTGAACACGCTGCTTTGGAACCAAGATTCTT

[0298] GGGCGGTTTCGCTATCATCACAAAGTCTTTCGCTCGTATCCATGAAACTAACTTGAAAAA

[0299] ACAAGGTCTATTGCCATTGAACTTCAAGAACCCAGCTGACTATGACAAGATCAACCCTG

[0300] ATGACAGAATCGATATTCTGGGTCTAGCTGAATTGGCTCCAGGTAAGCCTGTAACAATGA

[0301] GAGTTCATCCAAAGAATGGTAAGCCATGGGATGCTGTGTTGACCCATACTTTCAACGATG

[0302] AGCAAATTGAATGGTTCAAATATGGTTCTGCCTTAAATAAAATTAAGGCCGATGAGAAGAAATAA(SEQ ID NO: 15)

[0303] Saccharomyces cerevisiae 2U shuttle plasmid sequence:

[0304]

[0305] TTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTG

[0306] AAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCC

[0307] CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC

[0308] CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAG

[0309] CTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC

[0310] ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG

[0311] TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG

[0312] GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG

[0313] TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG

[0314] GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC

[0315] GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC

[0316] GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC

[0317] CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT

[0318] GGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAG

[0319] CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG

[0320] TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAG

[0321] AAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAA

[0322] GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT

[0323] GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTT

[0324] AATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC

[0325] CCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG

[0326] ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG

[0327] AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG

[0328] TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT

[0329] TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC

[0330] CCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT

[0331] TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGG

[0332] CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA

[0333] GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG

[0334] CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAA

[0335] AACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATG

[0336] TAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG

[0337] TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA

[0338] TGTTGAATACTCATACTCTTCCTTTTTCAATGGGTAATAACTGATATAATTAAATTGAAGCT

[0339] CTAATTTGTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGT

[0340] CAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCAC

[0341] GAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACG

[0342] CTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAG

[0343] CGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCC

[0344] TCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTG

[0345] ATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCG

[0346] CTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCA

[0347] GCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGAC

[0348] AGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGAC

[0349] AACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCT

[0350] GCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCG

[0351] GGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTG

[0352] TGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATC

[0353] CATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTT

[0354] AAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAA

[0355] GTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTC

[0356] AAGGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAAAGCTAGCT

[0357] TATCGATGATAAGCTGTCAAACATGAGAATTAATTCCACGGACTATAGACTATACCTAGTA

[0358] TACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTAC

[0359] CACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCT

[0360] TCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTT

[0361] CTACAATGGCTGCCATTCATTATTACCGATGTGACGCTGCAGCTTCCAATGATATTCGAA

[0362] TACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTA

[0363] CTTGTTACCCATCATTGATTTGAACATCCGAACCTGGAGTTTTCCCTGAAACAGATA

[0364] GTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATT

[0365] TCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCG

[0366] GTTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTG

[0367] TGCTTCATTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAAT

[0368] CTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAG

[0369] AATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACA

[0370] AAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCA

[0371] ACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCT

[0372] AACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGAT

[0373] AACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCT

[0374] CTTCCATAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGG

[0375] GTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCAT

[0376] ACTTTGTGAACAGAAAGTGATAGCGTTGATGATTTCATTGGTCAGAAAATTATGAACG

[0377] GTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTC

[0378] GATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATA

[0379] AACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATG

[0380] GGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGT

[0381] TTGTGGAAGCGGTATTCGCAATGGGAAGCTCCACCCCGGTTGATAATCAGAAAAGCCCC

[0382] AAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAGCGTTAATATTTTGTTAAT

[0383] TCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAAT

[0384] CCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACA

[0385] AGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA

[0386] GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCC

[0387] GTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCGATTTAGAGCTTGACGGGGAAA

[0388] GCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGG

[0389] CGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT (SEQ ID NO: 16)

[0390] Saccharomyces cerevisiae neomycin sulfate resistance gene sequence (including promoter, expression cassette, terminator):

[0391] GTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTG(SEQ IDNO:17)

[0392] Shuttle vector E. coli replicon sequence:

[0393] (SEQ ID NO: 18)

[0394] Shuttle vector ampicillin resistance gene expression cassette (including promoter, expression cassette, terminator):

[0395]

[0396] 酿酒酵母诱导型启动子GAL1序列:

[0397] ACGGATTAGAGCCGCCGAGCGACGAGCGAGCGGAGCGCAGCAGCGCCGAAGGAAGACTCTCCTCCGTGCGCTCCTCGTCTCGTCTTCACCGGTCGTCGTCCTGAAACGAGATGTGTGCCTCGCGCCGCACTGCTCCGAACAATAATACAATAAAGATTCTACAATACTAGCTATGGTTATGGTTATGAAGAGAAAAATGGCAGTACCTGGCCCCAAACCTCAAATGAACGAATCAATTAACAACCATAGGATGATGATAATGCGATTAGTTAGCTTATTCTATTTCTGGGGTAATTAATCAGCGAAGCGATGATGATTTTGATCTATTACAGATATATAATGCAAAACTGCATAACCACTTAACTAACTAATATAATACTTTCAACATTTTCGTGTTGATTACTTCTTATCAAATGTAATACAAAAATTGTAATATATACCTATACTACTACTACTTAACGTCAAGGAGACC(SEQ ID NO:20)

[0398] 酵母终止子CYC1序列:

[0399] ATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCT(SEQ ID NO:21)

[0400] 酿酒酵母组成型启动子TEF1序列:

[0401] CCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAA AAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG(SEQ ID NO:22)

[0402] 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

[0403] Figure 1 This is the interference result diagram of Example 4.

[0404] Figure 2 This is the interference result diagram of Example 5.

[0405] Figure 3 This is the interference result diagram of Example 6.

[0406] Figure 4 It is a schematic diagram of the principle of the present invention. DETAILED DESCRIPTION

[0407] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0408] 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.

[0409] 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)

[0410] 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.

[0411] Instruments and equipment:

[0412] 1.PCR instrument

[0413] 2. Electrophoresis apparatus

[0414] 3. Electrophoresis tank

[0415] 4. Ultra-low temperature refrigerator

[0416] 5. Biochemical incubator

[0417] 6. Autoclave

[0418] 7. Water bath

[0419] 8. Medical freezer

[0420] 9. Desktop Centrifuge

[0421] 10. Pipette

[0422] 11. Oven

[0423] 12.Electroshock converter and its matching 1mm / 2mm electroshock cup

[0424] Reagents:

[0425]

[0426]

[0427] Biomaterials:

[0428] E. coli related plasmids and primers:

[0429] Primers:

[0430] EP001:CAGCATCCTGCGATGCAGATCCGGAAC (SEQ ID NO: 23)

[0431] EP002:CAATCCGGATATAGTTCCTCCTTTCAGC (SEQ ID NO: 24)

[0432] EP003: GGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGC (SEQ ID NO: 25) EP004: CTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTAC (SEQ ID NO: 26) EP005: GGAAGGGAAGAAAGCGAAAGGAGC (SEQ ID NO: 27)

[0433] EP006:GGAACGTTGTGAGGGTAAACAACTGGC (SEQ ID NO: 28)

[0434] Gene sequences synthesized by the client:

[0435] CAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGAGAGCGTTCACCGACAAA

[0436] CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCGATGA

[0437] AGCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGCGAAGCCGCTAGCA

[0438] TTATACCTAGGACTGAGCTAGCTGTCAAGACTTCCGCGTTTCCAGACTTTACGAACAC

[0439] GGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCG

[0440] CTTCACGTTCGCTCGCGTATCGGTGATTCATTCGAAGAAAGGCCCACCCGTGAAGGTGA

[0441] GCCTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTC

[0442] GCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAA

[0443] AACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTT

[0444] CGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCG

[0445] GAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATA

[0446] TCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTA

[0447] TCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCA

[0448] AGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATT

[0449] TGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCG

[0450] TATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAG

[0451] TAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCT

[0452] CCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTT

[0453] CACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCG

[0454] GTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGAT

[0455] GGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCA

[0456] TACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACT

[0457] GCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTC

[0458] TGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCA

[0459] CGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATT

[0460] TTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTC

[0461] CATCCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTA

[0462] AAGGCGAAGCCAAATCCCCCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTG(SEQ ID NO:29)

[0463] 谷氨酸棒杆菌相关质粒与引物:

[0464] 引物:

[0465] GP001:CCATGAACAGAAATCCCGTAAATGCATGCCGCTTCGCCTTCG(SEQ ID NO:30)GP002:GAGCAGTATTTTGCGCGCCCACCGCAGTAGGCGCAGCTGATGCGATC(SEQ ID NO:31)

[0466] GP003:GGATTTCTGTTCATGGGGGTAATG(SEQ ID NO:32)

[0467] GP004:GCGCGCAAAATACTGCTCAGACG(SEQ ID NO:33)

[0468] GP005:GCTTACAGACAAGCTGTGACCGTCTC(SEQ ID NO:34)

[0469] GP006:TTGTGGCCGACCTTGAAGAGCTGG(SEQ ID NO:35)

[0470] 委托合成的序列:

[0471] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACC

[0472] TGCATCGATTTATTAGGACAGCTTCACTGCCACATCGTTCACCTTTTCTTCGCAGCCTGC

[0473] GCGGAATTCGGATGGGGATGCGCCGGTGCACTTCTTGAACACGCGGGAGAAGTACAGC

[0474] TGATCATCGAAGCCCACGTTGCGGCCCACGGTTGCGATTGGCATGCGGGTGGTGGACAG

[0475] CAGCAGCTTTGCCTGGGAGATGCGCTGATCTTCGCGCCAGGACAGCACGGAGATGCCC

[0476] AGCTGCTGGCGGAACAGGTGGGACAGGCGGGATGGGGACAGGCACACGTGCTGTGCC

[0477] ACGGATGCGATATCGAAGTTGGAATCTGCCAGGTGATCGGAGATGTACTGGCATGCTTCG

[0478] CGCACGCGGTTATCCATTGGTGGGTGCAGGGATTCGTTGATTGCTTCCATGCGGCGCAGC

[0479] AGCAGCTGTTCCAGCAGGTTGATTGCCAGCAGTTCGGAGTAGCGGCCTTCGCCCTGGCC

[0480] TGCGTTGATGATCTGGCCGAACAGATCGGAGAAGTGTGGCTGGTGTGCTTCATCTGGGC

[0481] GGAAGAAGCCGGTGTTTGCGAAGATGGATGGCCAGTTCAGCCATTCGTGCCAGTATGCG

[0482] CGTGGGCGGAAGTACACCCACTGGTGGTACCATTCGCGTGCTTCTGGGTGGCGGCCGTA

[0483] GTGGTGGATTTCGCCTGGTGGGAACAGCAGGATATCGCCTGGGCGGCACACGAATTCGC

[0484] GGCCCTGGTTCTTCACCACGCCCTGGCCGCGGATGGTCAGGTTCAGGATGTAGCCCTTC

[0485] ATGCCCAGTGGGCGATCGATGAAGAAATCCAGGTAGCCGTTTGCTTCGATTGGGGTCAG

[0486] GCCTGCCACCAGGTGTGCGTTGAAGGAGTAGCCTGGCAGCAGTGGATCGTTCTGTGCTT

[0487] CTGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAG

[0488] ACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCT

[0489] TATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAA

[0490] AGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGC

[0491] TATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACT

[0492] CTCTACTGTTTCTCCATCCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATC

[0493] GTGGAGGCTTTCGGCCGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGC

[0494] GCAACGAGGAGCGTTACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCCCAAATAAAA

[0495] CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC

[0496] TCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCC

[0497] GGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCTTGACA

[0498] ATTAATCATCCGGCTCGTATAATGCCCAACGCAGGAACTGGTAACGCTCCTCGTTGCGCT

[0499] CGTACTCGATCTCAACGTTCTTAGCCAGTGCATCTGGGCGGCCGAAAGCCTCCACGATG

[0500] ACGGAGTGGTCAATGACCATCTCGGAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTT

[0501] TCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTA

[0502] ATTCCGCTAGATGACGTGCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCGTCTGAGCAGTATTTTGCGCGC(SEQ ID NO: 36)Plasmids and primers related to Saccharomyces cerevisiae:

[0503] Primers:

[0504] SP001: TGGCGAGAAAGGAAGGGAAGAAAGC(SEQ ID NO: 37)

[0505] SP002: TGGCCGATTCATTAATGCAGGGCC(SEQ ID NO: 38)

[0506] SP003: CCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT(SEQ ID NO: 39)SP004: GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGG(SEQ ID NO: 40)

[0507] SP005: CCGTAAAGCACTAAATCGGAACCC(SEQ ID NO: 41)

[0508] SP006:CTCACATGTTCTTTCCTGCGTTATCC(SEQ ID NO:42)

[0509] 委托合成的序列

[0510] TGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGA

[0511] GCTTGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAA

[0512] TGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAAC

[0513] GTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTA

[0514] ACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACAT

[0515] AACTAATTACATGATGTCAATTGGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTAT

[0516] GGCAGGTCGTCCATGGGAATTGAAGGCTCCCTTAGATTAGATTGCTATGCTTTCTTTCTAA

[0517] TGAGCAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAAACTTGAGA

[0518] AATTGAAGACCGTTTATTAACTTAAATATCAATGGGAGGTCATCGAAAGAGAAAAAAAT

[0519] CAAAAAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCCTTTTTCGA

[0520] CGAAGAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTACGGGTAAT

[0521] TAACGACACCCTAGAGGAAGAAAGAGGGGAAATTTAGTATGCTGTGCTTGGGTGTTTTG

[0522] AAGTGGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGA

[0523] AACATTTTGAAGCTATGGTGTGTGGTGTAGCGGTCACGCTGCGCGTAACCACCACACCC

[0524] GCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGTCAACTTTGTAT

[0525] AGAAAAGTTGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTC

[0526] TCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGC

[0527] CGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAA

[0528] AATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATA

[0529] GGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATG

[0530] ATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTT

[0531] TCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATT

[0532] GTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCGGTGGTTTAGGTCAATTGG

[0533] CTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATGGCAGGTCGTCCATGGGAATTGA

[0534] AGGCTCCAAAGATCTTAGGATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCC

[0535] CCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTAT

[0536] TTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTT

[0537] CTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTG

[0538] GGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCA(SEQ ID NO:43)

[0539] Example 1 Construction of the Escherichia coli Arabinose Promoter Plasmid Elimination Module

[0540] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0541] 1. The sequences SEQ ID NO: 29 synthesized by amplifying primers EP001 and EP002 according to the following system and PCR program, and primers EP003 and EP004 were used to amplify the plasmid template pet28a plasmid according to the same system and PCR program.

[0542] System:

[0543]

[0544]

[0545] PCR program:

[0546]

[0547] 2. Assemble the SanPrep Column PCR Product Purification Kit using a ready-to-use seamless cloning kit, and purify the PCR products EP001-EP002 and EP003-EP004 according to the purification kit instructions.

[0548] 3. Connect the two purified product fragments EP001-EP002 / EP003-EP004 according to the operation procedure shown in the ready-to-use seamless cloning kit instructions.

[0549] 4. Transform according to the following steps After overnight culture of 5α Chemically Competent Cells:

[0550] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", gently mix (gently pipette up and down or flick the tube wall several times), and let it stand on ice for 5 min.

[0551] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[0552] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C and 200 rpm for 40 min.

[0553] 4) Take an appropriate volume of the recovered solution and evenly spread it on an LB solid medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator overnight.

[0554] overnight.

[0555] (2) Sequencing verification experiment for the construction result:

[0556] Take the single colonies obtained from the spread plate and send them for sequencing verification of the construction result using the primers EP005 and EP006.

[0557] Plasmid construction result:

[0558] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT

[0559] TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG

[0560] GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA

[0561] CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCT

[0562] ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT

[0563] TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCAC

[0564] TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

[0565] TATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA

[0566] TTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA

[0567] ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC

[0568] GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA

[0569] AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC

[0570] AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAA

[0571] CCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

[0572] CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAA

[0573] TATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGC

[0574] AGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG

[0575] GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT

[0576] ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGAT

[0577] TGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC

[0578] ATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA

[0579] CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAA

[0580] CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0581] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

[0582] CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC

[0583] AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

[0584] AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

[0585] GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

[0586] GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

[0587] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

[0588] AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0589] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

[0590] CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA

[0591] CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

[0592] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

[0593] ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0594] GAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATG

[0595] GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTAT

[0596] CGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC

[0597] TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG

[0598] CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA

[0599] GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCT

[0600] CGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGG

[0601] CGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG

[0602] TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT

[0603] GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0604] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCA

[0605] CAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGAGAGC

[0606] GTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTT

[0607] TTATTTGCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGCG

[0608] AAGCCGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAGACTTCCGCGTTTCCAGACT

[0609] TTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGC

[0610] AGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCGAAGAAAGGCCCACC

[0611] CGTGAAGGTGAGCCTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCG

[0612] GCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAG

[0613] TTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCA

[0614] AAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCT

[0615] AACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCG

[0616] ACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCG

[0617] CGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGT

[0618] AACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCG

[0619] GCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCG

[0620] AAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGC

[0621] GCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAG

[0622] TGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCG

[0623] TCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCAT

[0624] TCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAAC

[0625] CCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCA

[0626] GCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGA

[0627] CATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTT

[0628] ATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAA

[0629] GTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCT

[0630] ATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACT

[0631] CTCTACTGTTTCTCCATCCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCT

[0632] GGCTGCTATCGCTAAAGGCGAAGCCAAATCCCCCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO: 44) The antisense RNA sequence:

[0633] GGCTTCGCCTTTAGCGATAGCAGCCAGGAAGCCTGCTTTGACATAGGCGGCTTCATC G(SEQ IDNO: 45)

[0634] Antisense RNA neutralizing sequence:

[0635] CCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGCGAAGCCAAATCCCC(SEQ ID NO: 46)

[0636] (3) Plasmid extraction experiment:

[0637] Select the available single colonies on the transformation plate in (1) according to the sequencing alignment results in (2), inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin for overnight activation, and complete the plasmid extraction according to the instructions of the Tiangen Plasmid Mini Kit.

[0638] Example 2 Construction of Arabinose Promoter Plasmid Elimination Module in Corynebacterium glutamicum

[0639] (1) High-fidelity Enzyme Amplification and Plasmid Construction Experiment:

[0640] 1. Design primers GP001 and GP002 to amplify the synthesized fragment (SEQ ID NO: 36) according to the following system and PCR program; design primers GP003 and GP004 to amplify the pEC-XK99E plasmid according to the following system and PCR program.

[0641] System:

[0642]

[0643]

[0644] PCR Program:

[0645]

[0646] 2. Use a 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 purification kit instructions.

[0647] 3. Connect the purified products GP001-GP002 / GP003-GP004 according to the operation procedure shown in the ready-to-use seamless cloning kit instructions.

[0648] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[0649] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (pipette gently or flick the tube wall several times), and let stand on ice for 5 min.

[0650] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0651] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C and 200 rpm for 40 min.

[0652] 4) Take an appropriate volume of the recovery solution and spread it evenly on an LB solid medium containing 50 μg / mL kanamycin, and incubate in an inverted 37 °C incubator overnight.

[0653] (2) Sequencing Verification Experiment for Construction Results:

[0654] The single colonies obtained from the coated plates were sent for sequencing verification of the construction results using the GP005 / GP006 primers.

[0655] Plasmid construction results:

[0656] CCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATCGTGGAGGCTTTCGGCCGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGCGCAACGAGGAGCGTTACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCCCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCTTGACAATTAATCATCCGGCTC

[0657] GTATAATGCCCAACGCAGGAACTGGTAACGCTCCTCGTTGCGCTCGTACTCGATCTCAAC

[0658] GTTCTTAGCCAGTGCATCTGGGCGGCCGAAAGCCTCCACGATGACGGAGTGGTCAATGA

[0659] CCATCTCGGAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTG

[0660] TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCCGCTAGATGACGT

[0661] GCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGCGCTCGCGGCTGGCTGCGGCAC

[0662] GACACGCGTCTGAGCAGTATTTTGCGCGCCCACCGCAGTAGGCGCAGCTGATGCGATCC

[0663] TCCACTACTGCGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCGGCCAGCTCTTCA

[0664] AGGTCGGCCACAAGCGTTTCTAGGTCGCTCGCGGCACTTGCCCAGTCGCGTGATGCTGG

[0665] CGCGTCTGTCGTATCGAGGGCGCGGAAAAATCCGATCACCGTTTTTAAATCGACGGCGG

[0666] CATCGAGTGCGTCGGACTCCAGCGCGACATCGGAGAGATCCACCGCTGATGCTTCAGGC

[0667] CAGTTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTTAAAAAT

[0668] TCTAGCCCCAATTCTGATAATTTCTTCCGGCACTCCTGCGAAAACCTGCGAGACTTCTTG

[0669] CCCAGAAAAAACGCCAAGCGCAGCGGTTACCGCACTTTTTTTCCAGGTGATTTCACCCT

[0670] GACCAGCGAAGCGGCACTTTAGTGCATGAGGTGTGCCCCTGGTTTCCCCTCTTTGGAGG

[0671] GTTCAACCCAAAAAAGCACACAAGCAAAAATGAAAATCATCATGAGCAAGTTGGTGCG

[0672] AAGCAGCAACGCGCTAGCTCCAAAAAGGTCTCCAGGATCTCGAGGAGATTTTTGAGGG

[0673] GGAGGGAGTCGAGGAAGAGCCAGAGCAGAAGGCGGGGAACCGTTCTCTGCCGACAGC

[0674] GTGAGCCCCCCTTAAAAATCAGGCCGGGGAGGAACCGGGGAGGGATCAGAGCTAGGAG

[0675] CGAGACACCCTAAAGGGGGGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTC

[0676] AGCCCGTGGATAGCGGAGGGTGAGGGCAAGTGAGAGCCAGAGCAAGGACGGGACCCC

[0677] TAAAGGGGGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGAC

[0678] GGCCGCGTTTAGCTTCCATTCCAAGTGCCTTTCTGACTTGTTGGATGCGCCTTTCACTGA

[0679] CACCTAGTTCGCCTGCAAGCTCACGAGTCGAGGGATCAGCAACCGATTGAGAACGGGC

[0680] ATCCAGGATCGCAGTTTTGACGCGAAGTTCGAGCAACTCGCCTGTCATTTCTCGGCGTTT

[0681] GTTTGCTTCCGCTAATCGCTGTCGCGTCTCCTGCGCATACTTACTTTCTGGGTCAGCCCAT

[0682] CTGCGTGCATTCGATGTAGCTGCGCCCCGTCGCCCCATCGTCGCTAGAGCTTTCCGCCCT

[0683] CGGCTGCTCTGCGTTTCCACCCGACGAGCAGGGACGACTGGCTGGCCTTTAGCCACGTA

[0684] GCCGCGCACACGACGCGCCATCGTCAGGCGATCACGCATGGCGGGAAGATCCGGCTCC

[0685] CGGCCGTCTGCACCGACCGCCTGGGCAACGTTGTACGCCACTTCATACGCGTCGATGAT

[0686] CTTGGCATCTTTTAGGCGCTCACCAGCAGCTTTGAGCTGGTATCCCACGGTCAACGCGT

[0687] GGCGAAACGCGGTCTCGTCGCGCGCTCGCTCTGGATTTGTCCAGAGCACTCGCACGCCG

[0688] TCGATCAGGTCGCCGGACGCGTCCAGGGCGCTCGGCAGGCTCGCGTCCAAAATCGCTA

[0689] GCGCCTTGGCTTCTGCGGTGGCGCGTTGTGCCGCTTCAATGCGGGCGCGTCCGCTGGAA

[0690] AAGTCCTGCTCAATGTACTTTTTCGGCTTCTGTGATCCGGTCATCGTTCGAGCAATCTCCA

[0691] TTAGGTCGGCCAGCCGATCCACACGATCATGCTGGCAGTGCCATTTATAGGCTGTCGGAT

[0692] CGTCTGAGACGTGCAGCGGCCACCGGCTCAGCCTATGCGAAAAAGCCTGGTCAGCGCC

[0693] GAAAACACGAGTCATTTCTTCCGTCGTTGCAGCCAGCAGGCGCATATTTGGGCTGGTTTT

[0694] ACCTGCTGCGGCATACACCGGGTCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAGCG

[0695] GATTCACGCCGATCCAAGCCGGCGCTTTTTCTAGGCGTGCCCATTTCTCTAAAATCGCGT

[0696] AGACCTGCGGGTTTACGTGCTCAATCTTCCCGCCGGCCTGGTGGCTGGGCACATCGATG

[0697] TCAAGCACGATCACCGCGGCATGTTGCGCGTGCGTCAGCGCAACGTACTGGCACCGCGT

[0698] CAGCGCTTTTGAGCCAGCCCGGTAGAGCTTTGGTTGGGTTTCGCCGGTATCCGGGTTTTT

[0699] AATCCAGGCGCTCGCGAAATCTCTTGTCTTGCTGCCCTGGAAGCTTTCGCGTCCCAGGT

[0700] GAGCGAGCAGTTCGCGGCGATCTTCTGCCGTCCAGCCGCGTGAGCCGCAGCGCATAGCT

[0701] TCGGGGTGGGTGTCGAACAGATCGGCGGACAATTTCCACGCGCTAGCTGTGACTGTGTC

[0702] CTGCGGATCGGCTAGAGTCATGTCTTGAGTGCTTTCTCCCAGCTGATGACTGGGGGTTAG

[0703] CCGACGCCCTGTGAGTTCCCGCTCACGGGGCGTTCAACTTTTTCAGGTATTTGTGCAGCT

[0704] TATCGTGTTTTCTTCGTAAATGAACGCTTAACTACCTTGTTAAACGTGGCAAATAGGCAG

[0705] GATTGATGGGGATCTAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAA

[0706] GGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGT

[0707] CAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCT

[0708] TGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACC

[0709] GGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGG

[0710] ATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGAC

[0711] AGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCG

[0712] CTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGAT

[0713] GCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCT

[0714] GTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACG

[0715] ACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGC

[0716] TGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAG

[0717] AAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGC

[0718] CCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCG

[0719] GTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTG

[0720] TTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGA

[0721] TGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGG

[0722] CCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTG

[0723] AAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCC

[0724] GATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGG

[0725] GGTTCGCGGAATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAG

[0726] ACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT

[0727] GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT

[0728] ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCC

[0729] TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACC

[0730] TCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG

[0731] GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGG

[0732] TTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC

[0733] GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT

[0734] AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG

[0735] GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC

[0736] TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT

[0737] GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT

[0738] AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG

[0739] CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC

[0740] ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGC

[0741] ATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGA

[0742] CACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA

[0743] CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC

[0744] CGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTAC

[0745] GGGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCAC

[0746] CTGCATCGATTTATTAGGACAGCTTCACTGCCACATCGTTCACCTTTTCTTCGCAGCCTGC

[0747] GCGGAATTCGGATGGGGATGCGCCGGTGCACTTCTTGAACACGCGGGAGAAGTACAGC

[0748] TGATCATCGAAGCCCACGTTGCGGCCCACGGTTGCGATTGGCATGCGGGTGGTGGACAG

[0749] CAGCAGCTTTGCCTGGGAGATGCGCTGATCTTCGCGCCAGGACAGCACGGAGATGCCC

[0750] AGCTGCTGGCGGAACAGGTGGGACAGGCGGGATGGGGACAGGCACACGTGCTGTGCC

[0751] ACGGATGCGATATCGAAGTTGGAATCTGCCAGGTGATCGGAGATGTACTGGCATGCTTCG

[0752] CGCACGCGGTTATCCATTGGTGGGTGCAGGGATTCGTTGATTGCTTCCATGCGGCGCAGC

[0753] AGCAGCTGTTCCAGCAGGTTGATTGCCAGCAGTTCGGAGTAGCGGCCTTCGCCCTGGCC

[0754] TGCGTTGATGATCTGGCCGAACAGATCGGAGAAGTGTGGCTGGTGTGCTTCATCTGGGC

[0755] GGAAGAAGCCGGTGTTTGCGAAGATGGATGGCCAGTTCAGCCATTCGTGCCAGTATGCG

[0756] CGTGGGCGGAAGTACACCCACTGGTGGTACCATTCGCGTGCTTCTGGGTGGCGGCCGTA

[0757] GTGGTGGATTTCGCCTGGTGGGAACAGCAGGATATCGCCTGGGCGGCACACGAATTCGC

[0758] GGCCCTGGTTCTTCACCACGCCCTGGCCGCGGATGGTCAGGTTCAGGATGTAGCCCTTC

[0759] ATGCCCAGTGGGCGATCGATGAAGAAATCCAGGTAGCCGTTTGCTTCGATTGGGGTCAG

[0760] GCCTGCCACCAGGTGTGCGTTGAAGGAGTAGCCTGGCAGCAGTGGATCGTTCTGTGCTT

[0761] CTGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAG

[0762] ACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCT

[0763] TATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAA

[0764] AGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGC

[0765] TATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT(SEQ ID NO:47)

[0766] The antisense RNA sequence therein:

[0767] CCCAACGCAGGAACTGGTAACGCTCCTCGTTGCGCTCGTACTCGATCTCAACGTTC TTAGCCAGTGCATCTGGGCGGCCGAAAGCCTCCACGATGACGGAGTGGTCAATGACCAT CTCGG(SEQ ID NO: 48)

[0768] Antisense RNA neutralizing sequence:

[0769] CCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATCGTGGAGGCTTTCG GCCGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGCGCAACGAGGAGCG TTACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCC(SEQ ID NO: 49)

[0770] (3) Plasmid extraction experiment:

[0771] Select available single colonies on the transformation plate in (1) according to the sequencing alignment results in (2), inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin for overnight activation, and complete plasmid extraction according to the instructions of the Tiangen plasmid mini extraction kit.

[0772] Example 3 Construction of an inducible GAL1 promoter plasmid elimination module in Saccharomyces cerevisiae

[0773] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0774] 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 amplify the yeast 2μm plasmid.

[0775] System:

[0776]

[0777] PCR program:

[0778]

[0779] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products SP001 - SP002 / SP003 - SP004 according to the instructions of the purification kit.

[0780] 3. Connect the purified products SP001 - SP002 / SP003 - SP004 respectively according to the operation procedures shown in the instruction manual of the ready - to - use seamless cloning kit.

[0781] 4. Transform according to the following steps After overnight culture of 5α Chemically Competent Cell:

[0782] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (pipette gently or flick the tube wall several times), and let it stand on ice for 5 min.

[0783] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[0784] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C, 200 rpm for 40 min.

[0785] 4) Take an appropriate volume of the recovered solution and spread it evenly on an LB solid medium containing 100 μg / mL AMP, and incubate it upside - down in a 37 °C incubator overnight.

[0786] (2) Sequencing verification experiment for the construction results:

[0787] Take single colonies obtained from two coated plates and use SP003 / SP004 primers to send them for sequencing to verify the construction results respectively, and obtain plasmids with correct sequencing.

[0788] Plasmid construction results:

[0789] CAACTTTGTATAGAAAAGTTGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAG

[0790] ATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTT

[0791] ATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAA

[0792] ATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATC

[0793] AGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACT

[0794] TTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTAT

[0795] CAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCGGTGGTT

[0796] TAGGTCAATTGGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATGGCAGGTCGTC

[0797] CATGGGAATTGAAGGCTCCAAAGATCTTAGGATCATGTAATTAGTTATGTCACGCTTACAT

[0798] TCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAG

[0799] TCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAA

[0800] TTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCT

[0801] TGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATG

[0802] AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC

[0803] TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAG

[0804] GCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA

[0805] AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAG

[0806] GCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC

[0807] CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC

[0808] TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC

[0809] GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT

[0810] GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC

[0811] GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAAC

[0812] AGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA

[0813] CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT

[0814] CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT

[0815] TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT

[0816] GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGG

[0817] TCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAA

[0818] ATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG

[0819] GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT

[0820] AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA

[0821] GACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCG

[0822] AGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGG

[0823] AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG

[0824] GCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGAT

[0825] CAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT

[0826] CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG

[0827] CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA

[0828] CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATAC

[0829] GGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTT

[0830] CGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT

[0831] CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA

[0832] ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC

[0833] TCATACTCTTCCTTTTTCAATGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTG

[0834] AGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTCAAGAAGGC

[0835] GATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCG

[0836] GTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTG

[0837] ATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTT

[0838] CCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCG

[0839] GGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTC

[0840] GTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCG

[0841] ATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCA

[0842] TTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCC

[0843] TGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAG

[0844] CACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTT

[0845] GCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTG

[0846] CGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCAT

[0847] AGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCA

[0848] ATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTG

[0849] GGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTC

[0850] CTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTCAAGGAAACCG

[0851] AAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAAAGCTAGCTTATCGATGATA

[0852] AGCTGTCAAACATGAGAATTAATTCCACGGACTATAGACTATACCTAGTATACTCCGTCTA

[0853] CTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTACCACTCTTTTGTT

[0854] ACTCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCTTCGCGATGTAG

[0855] TAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTTCTACAATGGCT

[0856] GCCATCATTATTATCCGATGTGACGCTGCAGCTTCTCAATGATATTCGAATACGCTTTGAG

[0857] GAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTACTTGTTACCCAT

[0858] CATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATAGTATATTTGAAC

[0859] CTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATTTCGGTTCCTGGA

[0860] GAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCGGTTCATTTTCTGC

[0861] GTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTGTGCTTCATTTTGT

[0862] AGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTT

[0863] TTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCA

[0864] TTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGC

[0865] TGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTAT

[0866] ACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTT

[0867] AGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTG

[0868] TAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAA

[0869] GCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAA

[0870] GATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGA

[0871] AAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTG

[0872] TCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAA

[0873] TAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTA

[0874] GAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGG

[0875] ATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTAT

[0876] TCGCAATGGGAAGCTCCACCCCGGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGAT

[0877] TGTATAAGCAAATATTTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTT

[0878] GTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAA

[0879] AAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTA

[0880] AAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCAC

[0881] TACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATC

[0882] GGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGC

[0883] GAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGAGCTT

[0884] GCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTT

[0885] ACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTC

[0886] TTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTC

[0887] CTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACT

[0888] AATTACATGATGTCAATTGGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATGGCA

[0889] GGTCGTCCATGGGAATTGAAGGCTCCCTTAGATTAGATTGCTATGCTTTCTTTCTAATGAG

[0890] CAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAAACTTGAGAAATT

[0891] GAAGACCGTTTATTAACTTAAATATCAATGGGAGGTCATCGAAAGAGAAAAAAATCAAA

[0892] AAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCCTTTTTCGACGAA

[0893] GAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTACGGGTAATTAAC

[0894] GACACCCTAGAGGAAGAAAGAGGGGAAATTTAGTATGCTGTGCTTGGGTGTTTTGAAGT

[0895] GGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGAAACA

[0896] The antisense RNA sequence involved in TTTTGAAGCTATGGTGTGTGGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT (SEQ ID NO: 50):

[0897] GGAGCCTTCAATTCCCATGGACGACCTGCCATAACATCAACGGCATCAGCACCACCAACACCAATAGCCAATTGAC (SEQ ID NO: 51)

[0898] Antisense RNA neutralizing sequence:

[0899] GGTGGTTTAGGTCAATTGGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATG GCAGGTCGTCCATGGGAATTGAAGGCTCCAAAGATCTTAGG. (SEQ ID NO: 52)

[0900] (3) Plasmid extraction experiment:

[0901] Select available single colonies on the transformation plate in (1) according to the sequencing alignment results in (2), inoculate them into 5 mL of LB liquid medium with 100 μg / mL AMP for overnight activation, and complete plasmid extraction according to the instructions of the Tiangen plasmid miniprep kit.

[0902] Example 4 Use the Escherichia coli cascade reaction plasmid elimination module to eliminate plasmids

[0903] 1. Prepare the Escherichia coli to be transformed into competent cells according to the procedure described in the super competent cell preparation kit.

[0904] 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 pipette or flick the tube wall several times), and let it stand on ice for 5 min.

[0905] 3. Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let it stand for 2 min (do not shake the sample during the standing on ice process, otherwise the transformation efficiency will be reduced).

[0906] 4. Add 700 μL of sterile liquid medium (SOB or LB) without antibiotics but containing 1% arabinose to a centrifuge tube and mix well.

[0907] 5. Take an appropriate volume of the resuscitation solution and evenly spread it on an LB solid medium containing 50 μg / mL kanamycin and 1% arabinose, and incubate it upside down in a 37 °C incubator overnight.

[0908] 6. Add the Escherichia coli constructed in "5" or the frozen bacteria / stab culture of Escherichia coli containing the construction module in "5" to an LB liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, and culture it at 37 °C and 220 rpm. After culturing overnight (12 h), streak it on an antibiotic-free LB solid medium plate.

[0909] 7. Take the single colonies obtained in "6" and spot them on both antibiotic-free and antibiotic-containing solid medium plates (each single colony needs to be spotted on the plate twice to ensure the distinction of phenotypic differences).

[0910] Specific results are as Figure 1 shown. After plasmid curing, most of the bacteria lost their resistance to antibiotics and were no longer able to grow on the antibiotic-containing plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully cured. Inoculating the corresponding successfully cured strains can obtain strains with successfully cured plasmids.

[0911] Example 5

[0912] 1. Overnight activate the Corynebacterium glutamicum ATCC13032 strain in 5 mL of brain heart infusion liquid medium.

[0913] 2. Inoculate all the activated Corynebacterium glutamicum bacterial solution into 45 mL of Epo liquid medium to make the final OD600 reach 0.2, and grow it at 200 rpm and 30 °C until the OD600 reaches 0.9.

[0914] 3. Transfer the bacterial solution to a sterile 50 mL centrifuge tube, centrifuge to remove the supernatant, wash the cells 4 times with ice-cold 10% sterilized glycerol, aliquot 100 μL per tube into sterile 1.5 mL centrifuge tubes, and store at 70 °C or use immediately.

[0915] 4. During electroporation, thaw the aliquoted competent cells on ice and add 5 μL of the plasmid obtained in the "Construction experiment of Corynebacterium glutamicum cascade reaction plasmid curing module".

[0916] 5. Transfer the mixture to a cold electroporation cuvette (0.1 cm), and perform a pulsed electric shock at 1.8 kV for 5 ms. Immediately after electroporation, add 1 mL of brain heart infusion medium containing 1% arabinose to a cuvette, mix well, and transfer it to a 1.5 mL centrifuge tube.

[0917] 6. Incubate for 1 hour at 30 °C.

[0918] 7. Finally, spread on a brain heart infusion agar plate containing 50 μg / mL kanamycin and 1% arabinose.

[0919] 8. Add the Corynebacterium glutamicum constructed in "7" or the frozen / punctured bacteria of Corynebacterium glutamicum containing the construction module in "7" to a brain heart infusion liquid medium without antibiotics with a volume not exceeding 1 / 5 of the conical flask volume, and culture overnight (18 h) at 30 °C and 220 rpm.

[0920] 9. Streak on a solid LB medium plate without antibiotics.

[0921] 10. Take the single colonies obtained in "9" and spot them on solid medium plates without antibiotics and with antibiotics respectively (each single colony needs to be spotted on the plate twice to ensure the distinction of phenotypic differences).

[0922] The specific results are as Figure 2 shown. After plasmid elimination, most of the bacteria lost their resistance to antibiotics and could no longer grow in the antibiotic-containing plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully eliminated. Inoculating the corresponding strains with successful elimination can obtain the strains with successfully eliminated plasmids.

[0923] Example 6 Plasmid elimination using the plasmid elimination module of Saccharomyces cerevisiae cascade reaction

[0924] First, prepare the following reagents:

[0925] 10*TE buffer

[0926] 100 mM LiAC buffer

[0927] 1 M DTT buffer

[0928] ddH2O

[0929] 1 M sorbitol solution

[0930] Preparation of Saccharomyces cerevisiae competent cells:

[0931] 1. Pre-cool the reagents.

[0932] 2. Pipette 30 μL from 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, and then pipette 100 μL from it and inoculate it into 100 mL of liquid YPD medium until the cell concentration OD600 grows to 1.3 - 1.5.

[0933] 3. Divide the bacterial solution into three pre-cooled 50 mL centrifuge tubes, centrifuge at 4 °C and 5000 r×min-1 for 5 min, discard the supernatant, and collect the bacterial cells.

[0934] 4. Resuspend the cells by pipetting up and down with 4 mL of pre-cooled ddH2O. 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 1 M DTT buffer. Mix well by pipetting. Incubate the centrifuge tube in a constant temperature water bath shaker at 30°C and 50 r×min-1 for 45 min.

[0935] 5. Add 13.5 mL of pre-cooled ddH2O to a 50 mL centrifuge tube. Centrifuge at 5000 r×min-1 for 5 min at 4°C. Discard the supernatant and collect the cells.

[0936] 6. Add 25 mL of pre-cooled ddH2O to a 50 mL centrifuge tube and resuspend the cells by pipetting. Centrifuge at 5000 r×min-1 for 5 min. Discard the supernatant and collect the cells. Then wash the cells twice with 1 mol·L-1 sorbitol (25 mL for the first time and 20 mL for the second time).

[0937] 7. Add 1 mL of pre-cooled sorbitol to the centrifuge tube to resuspend the cells, and aliquot them into 1.5 mL EP tubes, 80 μL per tube, for immediate use.

[0938] 8. During electroporation, place the aliquoted competent cells on ice and add 5 μL of the plasmid obtained from the "Construction Experiment of Saccharomyces cerevisiae Cascade Reaction Plasmid Elimination Module".

[0939] 9. Transfer the mixture to a cold electroporation cuvette (0.2 cm) and perform pulsed electroporation at 1.5 kV, 200 Ω, 25 μF, and 5 ms. Immediately after electroporation, add 1 mL of antibiotic-free YPD medium containing 2% galactose to the cuvette, mix well, and transfer it to a 1.5 mL centrifuge tube. Incubate in a shaker at 30°C and 200 rpm for 1 - 3 h.

[0940] 10. Centrifuge to collect the cells. Take 150 μL of the cell suspension and spread it on a resistance plate containing 500 μg / mL neomycin sulfate with 2% galactose, and culture at 30°C for 2 - 3 days.

[0941] 11. Add the cryopreserved bacteria or stab culture of Saccharomyces cerevisiae constructed in "10" or Saccharomyces cerevisiae containing the module constructed in "10" to an antibiotic-free YPD liquid medium with a volume not exceeding 1 / 5 of the conical flask volume. Culture at 30°C and 220 rpm for 48 h, and then streak on an antibiotic-free YPD solid medium plate.

[0942] Take the single colonies obtained in "11" and spot them on antibiotic-free and antibiotic-containing (500 μg / mL neomycin sulfate) YPD solid medium plates respectively (each single colony needs to be spotted twice to ensure the distinction of phenotypic differences).

[0943] The specific results are as Figure 3As shown, after plasmid elimination, most of the growing bacteria lost their antibiotic resistance and were no longer able to grow on antibiotic plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully eliminated. Inoculating the strain with successful elimination can obtain the strain with successfully eliminated plasmid.

[0944] The foregoing has shown and described 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. Any technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A plasmid elimination module based on cascade reaction, characterized in that, It includes three types of independent expression regions. One type of region is the antisense RNA expression region, which consists of an antisense RNA, a promoter of the antisense RNA, and a terminator; the antisense RNA is the antisense RNA of the mRNA of a key metabolic gene, and is an RNA fragment that can bind to the mRNA of the key metabolic gene and hinder the translation of the mRNA of the key metabolic gene; one type of region is the antisense neutralizing RNA expression region, which consists of an antisense neutralizing RNA, an inducible promoter of the antisense neutralizing RNA, and a terminator; the last type of region is the transcription factor region, which is composed of a constitutive promoter carried by a transcription factor fragment, and it includes an RNA polymerase binding site, 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, and is an RNA fragment that can bind to the antisense RNA so that it cannot bind to the mRNA of the key metabolic gene and inhibits the antisense RNA from causing translation hindrance to the mRNA of the key metabolic gene.

2. The plasmid elimination module according to claim 1, wherein The transcription factor is a protein that can induce the transcription of genes downstream of the binding site after binding to a specific compound; or a protein that can induce the transcription of downstream genes before binding to a specific compound and cannot induce the transcription of genes downstream of the binding site after binding to the specific compound.

3. The plasmid elimination module according to claim 2, characterized in that, The transcription factor is the arabinose transcription factor.

4. The plasmid elimination module according to claim 1, wherein The constitutive promoter carried by the transcription factor fragment is the constitutive promoter carried by the arabinose transcription factor.

5. The plasmid elimination module according to claim 1, characterized in that The antisense RNA promoter includes the J23119 promoter, T7, tet, trc, trp, tac, and constitutive promoters related to the common antibiotics chloramphenicol, kanamycin, and penicillin resistance genes of Bacillus glutamicum, or the TEF1 promoter, and the antisense RNA terminator includes the Cyc1 terminator, the rrnB T1 terminator, or the rrnB T2 terminator.

6. The plasmid elimination module according to claim 1, wherein The inducible promoter of the antisense neutralizing RNA includes the GAL1 promoter, the arabinose promoter, and the antisense RNA terminator includes the Cyc1 terminator, the rrnB T1 terminator, or the T7 terminator.

7. The plasmid elimination module according to claim 1, characterized in that The plasmid elimination module is carried on the plasmid to be eliminated.

8. The plasmid elimination module according to claim 1, wherein The plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and the yeast 2μ plasmid.

9. The plasmid elimination module according to claim 1, wherein The key metabolic genes described above are genes related to the TCA cycle, protein synthesis, aerobic respiration, anaerobic respiration, lipid synthesis, cell wall synthesis, sugar transport pathways, amino acid transport pathways, ATP synthesis, cytoskeleton synthesis, 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 isozyme genes of the above genes.

10. The plasmid elimination module according to any one of claims 1 to 9, characterized in that, The elimination target of the plasmid elimination module is all microorganisms, animal or plant cells that have stable free plasmids, there is a certain probability that the free plasmids will not be distributed to daughter cells, and the cells will not die or be unable to proliferate after the free plasmids are eliminated.

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