Repression protein-based plasmid elimination module

Abstract

The invention relates to the field of gene engineering, in particular to a plasmid elimination module based on repressor protein, which comprises two types of mutually independent expression regions, one type of region is a repressor protein gene transcription region, and consists of a constitutive promoter carried by a repressor protein gene and a recombinant plasmid. Comprising an RNA polymerase binding site, a transcription start site, a ribosome binding site, a repressor protein translation region and a terminator. The other region is an antisense RNA expression region which is composed of a constitutive promoter, a repressor protein binding site, antisense RNA and a terminator; the antisense RNA is the antisense RNA of a metabolic key gene mRNA (messenger Ribonucleic Acid). 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 repressor protein. 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 genome editing or gene circuit plasmids to achieve genome editing or gene circuit design purposes, the genome editing tools or gene circuits carried on the plasmids become unstable factors in the cell genome. At this time, it is necessary to eliminate the plasmids 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, and it is prone to failure.

[0005] The plasmid elimination technique based on CRISPR proteins can eliminate multiple plasmids simultaneously and freely select the types of plasmids to be eliminated. However, there are risks of CRISPR protein gene mutation failure or CRISPR protein cleavage off-target, and the gene sequences of CRISPR-related protein elements are 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 cells, 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 technique based on antisense RNA and designs a plasmid elimination module. This module can promote the loss of episomal plasmids with a relatively low spontaneous loss rate in cells, can efficiently achieve the selective elimination or synchronous elimination of multiple episomal plasmids, can be applied to the selective elimination or synchronous elimination of different types (copy numbers) of episomal plasmids in eukaryotic and prokaryotic cells, and has better tolerance to sequence mutations than the plasmid elimination technique based on CRISPR proteins. The gene sequences of related protein elements are shorter than those of CRISPR and are 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 prerequisites are met, the plasmid elimination module can be used for the elimination of constructed plasmids after the construction of genetically engineered bacteria, the elimination of artificially constructed plasmids, and can also be used in conjunction with sequence editing tools to achieve the elimination of natural plasmids in cells.

[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 repressor protein comprises two types of independent expression regions, one of which is the repressor protein gene transcription region, which is composed of the constitutive promoter of the repressor protein gene, including its RNA polymerase binding site and transcription start site, ribosome binding site, repressor protein translation region, and terminator; the other type of region is the antisense RNA expression region, which is composed of a constitutive promoter, a repressor protein binding site, antisense RNA and a terminator; 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.

[0012] According to an embodiment of the present invention, the repressor protein is a repressor protein that can bind to a specific DNA sequence, inhibit downstream DNA transcription after binding, can be activated or deactivated by a compound, and does not affect the growth of chassis cells. The repressor protein binding site binds to the repressor protein and prevents RNA polymerization from passing through or crossing the binding site to transcribe the DNA sequence downstream of the binding site.

[0013] According to an embodiment of the present invention, the repressor protein gene is a LacI protein gene, and the repressor protein binding site is a LacI protein binding site.

[0014] According to an embodiment of the present invention, the constitutive promoter is a specific DNA sequence that can bind RNA polymerase with a certain intensity in a cell and initiate the transcription function of RNA polymerase to transcribe the downstream functional sequence and antisense RNA.

[0015] According to an embodiment of the present invention, the constitutive promoters include T7, tet, trc, trp, J23119 promoter, tac, and constitutive promoters related to common antibiotics chloramphenicol, kanamycin, and penicillin resistance genes of Corynebacterium glutamicum.

[0016] According to an embodiment of the present invention, the plasmid elimination module is carried on the plasmid to be eliminated.

[0017] According to an embodiment of the present invention, the plasmid to be eliminated is a circular or linear DNA that can replicate independently outside the genome stably in a cell, or replicate with the genome and be passed on to daughter cells.

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

[0019] According to an embodiment of the present invention, the gene for antisense RNA interference is the antisense RNA of the mRNA of a key metabolic gene, which can bind to the mRNA of the key metabolic gene and hinder the translation of the mRNA of the key metabolic gene.

[0020] 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.

[0021] According to an embodiment of the present invention, the elimination target of the plasmid elimination module is all microorganisms, animals, or plant cells that have stably free plasmids, have a probability of generating plasmid-free daughter cells during proliferation, and the elimination of free plasmids will not cause cell death or inability to proliferate.

[0022] For the convenience of those skilled in the art to understand the present invention, the main vocabulary explanations are as follows:

[0023] Genetic circuit: A combination of DNA sequences that arranges specific genetic elements such as promoters, terminators, protein translation regions, and specific protein-binding regions in a single-site or multi-site layout according to certain rules to achieve specific functions.

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

[0025] Programmable: The use of this module is not limited by the plasmid copy number, and selective elimination can be achieved in cells containing multiple types of 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 in the present invention.

[0028] Shorter required sequence: When using the cell's native regulatory factors, the total length of the plasmid elimination module proposed in 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 proposed in the present invention usually does not exceed 3 kbp.

[0029] Constructing plasmids: mainly refers to tool plasmids that carry 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 plasmids: mainly refer to plasmids that do not involve genome editing and are artificially constructed to express homologous / heterologous proteins or some functional genetic circuits.

[0031] Plasmid backbone: The DNA sequence carrying the plasmid replicon and plasmid maintenance genes.

[0032] Free plasmid: A linear or circular double-stranded DNA that can autonomously replicate in cells either independently or together with the genome, can be distributed to daughter cells during cell proliferation, and can stably exist outside the genome.

[0033] Constitutive promoter: A specific DNA sequence that can be recognized by the Escherichia coli σ factor and recruit RNA polymerase subunits for transcription of the downstream region.

[0034] Illustrated as follows

[0035] Escherichia coli-related sequences

[0036] Full sequence of the interference region of the Escherichia coli acnB gene:

[0037] ACTATGACAATGAGAGCGAGGAGAACCGTCGTGCTAGAAGAATACCGTAAGCACGTAG

[0038] CTGAGCGTGCCGCTGAGGGGATTGCGCCCAAACCCCTGGATGCAAACCAAATGGCCGC

[0039] ACTTGTAGAGCTGCTGAAAAACCCGCCCGCGGGCGAAGAAGAATTCCTGTTAGATCTGT

[0040] TAACCAACCGTGTTCCCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTG

[0041] GCTGCTATCGCTAAAGGCGAAGCCAAATCCCCTCTGCTGACTCCGGAAAAAGCCATCGA

[0042] ACTGCTGGGCACCATGCAGGGTGGTTACAACATTCATCCGCTGATCGACGCGCTGGATG

[0043] ATGCCAAACTGGCACCGATCGCTGCCAAAGCACTTTCTCACACACTGCTGATGTTCGAT

[0044] AACTTCTATGACGTAGAAGAGAAAGCGAAAGCAGGCAACGAATATGCGAAGCAGGTAA

[0045] TGCAGTCCTGGGCGGATGCCGAATGGTTCCTGAATCGCCCGGCGCTGGCTGAAAAACTG

[0046] ACCGTTACCGTCTTCAAAGTCACTGGCGAAACCAACACCGATGACCTCTCTCCGGCACC

[0047] GGATGCGTGGTCACGCCCGGATATCCCACTGCACGCGCTGGCGATGCTGAAAAACGCCC

[0048] GTGAAGGCATCGAGCCAGACCAGCCAGGTGTTGTTGGTCCGATCAAGCAAATCGAAGC

[0049] TCTGCAACAGAAAGGTTTCCCGCTGGCGTACGTCGGTGACGTTGTGGGTACGGGTTCAT

[0050] CGCGTAAATCCGCCACGAACTCCGTACTGTGGTTTATGGGTGATGATATTCCACATGTGC

[0051] CGAACAAACGCGGCGGTGGTTTGTGCCTCGGCGGTAAAATTGCACCAATCTTCTTTAAC

[0052] ACAATGGAAGACGCAGGTGCACTGCCAATCGAAGTCGACGTCTCTAACCTGAACATGG

[0053] GCGACGTAATTGACGTTTACCCGTACAAAGGTGAAGTGCGTAACCACGAAACCGGCGA

[0054] ACTGCTGGCGACCTTCGAACTGAAAACCGACGTGCTGATTGATGAAGTGCGTGCTGGC

[0055] GGCCGTATTCCGCTGATTATTGGGCGTGGCCTGACCACCAAAGCGCGTGAAGCACTTGG

[0056] TCTGCCGCACAGTGATGTGTTCCGTCAGGCGAAAGATGTCGCTGAGAGCGATCGCGGCT

[0057] TCTCGCTGGCGCAAAAAATGGTAGGCCGTGCCTGTGGCGTGAAAGGCATTCGTCCGGGC

[0058] GCGTACTGCGAACCGAAAATGACTTCTGTAGGCTCTCAGGACACCACCGGCCCGATGAC

[0059] CCGTGATGAACTGAAAGACCTGGCGTGCCTGGGCTTCTCGGCTGACCTGGTGATGCAGT

[0060] CTTTCTGCCACACCGCGGCGTATCCGAAGCCAGTTGACGTGAACACGCACCACACGCTG

[0061] CCGGACTTCATTATGAACCGTGGCGGTGTGTCGCTGCGTCCGGGTGACGGCGTCATTCA

[0062] CTCCTGGCTGAACCGTATGCTGCTGCCGGATACCGTCGGTACCGGTGGTGACTCCCATAC

[0063] CCGTTTCCCGATCGGTATCTCTTTCCCGGCGGGTTCTGGTCTGGTGGCGTTTGCTGCCGC

[0064] AACTGGCGTAATGCCGCTGGATATGCCGGAATCCGTTCTGGTGCGCTTCAAAGGCAAAA

[0065] TGCAGCCGGGCATCACCCTGCGCGATCTGGTACACGCGATCCCGCTGTATGCGATCAAA

[0066] CAAGGTCTGCTGACCGTTGAGAAGAAAGGCAAGAAAAACATCTTCTCTGGCCGCATCC

[0067] TGGAAATTGAAGGTCTGCCGGATCTGAAAGTTGAGCAGGCCTTTGAGCTAACCGATGCG

[0068] TCCGCCGAGCGTTCTGCCGCTGGTTGTACCATCAAGCTGAACAAAGAACCGATCATCGA

[0069] ATACCTGAACTCTAACATCGTCCTGCTGAAGTGGATGATCGCGGAAGGTTACGGCGATC

[0070] GTCGTACCCTGGAACGTCGTATTCAGGGCATGGAAAAATGGCTGGCGAATCCTGAGCTG

[0071] CTGGAAGCCGATGCAGATGCGGAATACGCGGCAGTGATCGACATCGATCTGGCGGATATT

[0072] AAAGAGCCAATCCTCTGTGCACCGAACGACCCGGACGACGCACGTCCGCTGTCTGCGG

[0073] TACAGGGCGAGAAGATCGACGAAGTGTTTATCGGTTCCTGCATGACCAACATCGGTCAC

[0074] TTCCGTGCTGCGGGTAAACTGCTGGATGCGCACAAAGGCCAGTTGCCGACGCGCCTGTG

[0075] GGTGGCACCGCCAACCCGTATGGATGCCGCGCAGTTGACCGAAGAAGGCTACTATAGCG

[0076] TCTTCGGTAAGAGCGGTGCGCGTATCGAGATCCCTGGCTGTTCCCTGTGTATGGGTAACC

[0077] AGGCGCGTGTAGCAGACGGTGCGACGGTGGTTTCCACCTCTACCCGTAACTTCCCGAAC

[0078] CGTCTGGGGACTGGCGCGAATGTCTTCCTGGCTTCTGCGGAACTGGCGGCTGTTGCGGC

[0079] GCTGATTGGCAAACTGCCGACGCCGGAAGAGTACCAGACCTACGTGGCGCAAGTAGAT

[0080] AAAACTGCCGTTGATACTTATCGTTATCTGAACTTCAACCAGCTTTCTCAGTACACCGAAAAAGCCGATGGGGTGATTTTCCAGACTGCGGTTTAA(SEQ ID NO:1)

[0081] Full sequence of pET28a plasmid:

[0082] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGC

[0083] GCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT

[0084] CCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAG

[0085] GGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTT

[0086] CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGT

[0087] TCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT

[0088] CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA

[0089] ACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTT

[0090] TCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT

[0091] CCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATT

[0092] CATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA

[0093] CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCG

[0094] TCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAA

[0095] ATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCA

[0096] GACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAAC

[0097] CGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC

[0098] AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATA

[0099] TTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAG

[0100] TGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGC

[0101] ATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTAC

[0102] CTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTG

[0103] TCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCAT

[0104] GTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACC

[0105] CCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACG

[0106] TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG

[0107] ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCG

[0108] GTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

[0109] CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA

[0110] GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC

[0111] CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG

[0112] CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA

[0113] CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

[0114] GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACG

[0115] AGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT

[0116] CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG

[0117] CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT

[0118] TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATAC

[0119] CGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA

[0120] GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGT

[0121] GCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATC

[0122] GCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCT

[0123] GACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGC

[0124] TGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAG

[0125] CTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTC

[0126] GTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGC

[0127] GGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGT

[0128] AATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATG

[0129] CCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCA

[0130] GAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCAC

[0131] AGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGAC

[0132] TTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCA

[0133] GGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT

[0134] CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACG

[0135] ATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACG

[0136] TTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACC

[0137] GCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGA

[0138] CCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGT

[0139] GCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTC

[0140] TCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGT

[0141] TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0142] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA

[0143] CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG

[0144] CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACG

[0145] GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACC

[0146] AACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG

[0147] CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0148] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTG

[0149] AGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC

[0150] TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTAC

[0151] CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAAT

[0152] AACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0153] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC

[0154] AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0155] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG

[0156] TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA

[0157] ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG

[0158] CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGAC

[0159] ATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT

[0160] CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0161] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG

[0162] CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGG

[0163] GCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGA

[0164] TCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGT

[0165] GATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAAT

[0166] ACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT

[0167] TAACTTTAAGAAGGAGATATACCATGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC

[0168] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:2)

[0169] T7 promoter sequence (arrow indicates the transcription start direction):

[0170] TAATACGACTCACTATAGG→(SEQ ID NO:3)

[0171] Tet promoter sequence:

[0172] TTGACAGCTTATCATCGATTAGCTTTAAT→(SEQ ID NO:4)

[0173] T7 terminator sequence (arrow indicates the transcription termination direction):

[0174] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO:5)LacI protein expression cassette (including promoter, CDS region, terminator):

[0175] CGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAG

[0176] CGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGAT

[0177] AAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAG

[0178] CTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAG

[0179] CTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTG

[0180] CCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCC

[0181] AGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTG

[0182] GCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCC

[0183] TGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCA

[0184] CTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCC

[0185] AGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCAT

[0186] TTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGG

[0187] CTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGA

[0188] CAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGC

[0189] TCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGG

[0190] TCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGG

[0191] CATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGA

[0192] TTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCAC

[0193] GCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGT

[0194] GCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTG

[0195] TTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCG

[0196] CGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG

[0197] ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAAT

[0198] TGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTC(SEQ IDNO:6)

[0199] LacI protein binding site sequence:

[0200] GGAATTGTGAGCGGATAACAATTCC(SEQ ID NO:7)

[0201] Corynebacterium glutamicum related sequence:

[0202] Full sequence of the CDS region of the Corynebacterium glutamicum acn gene:

[0203] TTGGAGCTCACTGTGACTGAAAGCAAGAACTCCTTCAATGCTAAGAGCACCCTTGAAGT

[0204] TGGCGACAAGTCCTATGACTACTTCGCCCTCTCTGCAGTGCCTGGCATGGAGAAGCTGC

[0205] CGTACTCCCTCAAGGTTCTCGGAGAGAACCTTCTTCGTACCGAAGACGGCGCAAACATC

[0206] ACCAACGAGCACATTGAGGCTATCGCCAACTGGGATGCATCTTCCGATCCAAGCATCGA

[0207] AATCCAGTTCACCCCAGCCCGTGTTCTCATGCAGGACTTCACCGGTGTCCCTTGTGTAGT

[0208] TGACCTCGCAACCATGCGTGAGGCAGTTGCTGCACTCGGTGGCGACCCTAACGACGTCA

[0209] ACCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATCGTGGAGGCTTTCGGC

[0210] CGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGCGCAACGAGGAGCGTT

[0211] ACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCCAACTTCCGCGTTGTTCCTCCAGGA

[0212] ACCGGTATCGTCCACCAGGTCAACATTGAGTACTTGGCTCGCGTCGTCTTCGACAACGA

[0213] GGGCCTTGCATACCCAGATACCTGCATCGGTACCGACTCCCACACCACCATGGAAAACG

[0214] GCCTGGGCATCCTGGGCTGGGGCGTTGGTGGCATTGAGGCTGAAGCAGCAATGCTCGG

[0215] CCAGCCAGTGTCCATGCTGATCCCTCGCGTTGTTGGCTTCAAGTTGACCGGCGAGATCC

[0216] CAGTAGGCGTTACCGCAACTGACGTTGTGCTGACCATCACCGAAATGCTGCGCGACCAC

[0217] GGCGTCGTCCAGAAGTTCGTTGAGTTCTACGGCTCCGGTGTTAAGGCTGTTCCACTGGC

[0218] TAACCGTGCAACCATCGGCAACATGTCCCCAGAGTTCGGCTCCACCTGTGCGATGTTCC

[0219] CAATCGACGAGGAGACCACCAAGTACCTGCGCCTCACCGGCCGCCCAGAAGAGCAGGT

[0220] TGCACTGGTCGAGGCTTACGCCAAGGCGCAGGGCATGTGGCTCGACGAGGACACCGTT

[0221] GAAGCTGAGTACTCCGAGTACCTCGAGCTGGACCTGTCCACCGTTGTTCCTTCCATCGC

[0222] TGGCCCTAAGCGCCCACAGGACCGCATCCTTCTCTCCGAGGCAAAGGAGCAGTTCCGTA

[0223] AGGATCTGCCAACCTACACCGACGACGCTGTTTCCGTAGACACCTCCATCCCTGCAACC

[0224] CGCATGGTTAACGAAGGTGGCGGACAGCCTGAAGGCGGCGTCGAAGCTGACAACTACA

[0225] ACGCTTCCTGGGCTGGCTCCGGCGAGTCCTTGGCTACTGGCGCAGAAGGACGTCCTTCC

[0226] AAGCCAGTCACCGTTGCATCCCCACAGGGTGGCGAGTACACCATCGACCACGGCATGGT

[0227] TGCAATTGCATCCATCACCTCTTGCACCAACACCTCTAACCCATCCGTGATGATCGGCGC

[0228] TGGCCTGATCGCACGTAAGGCAGCAGAAAAGGGCCTCAAGTCCAAGCCTTGGGTTAAG

[0229] ACCATCTGTGCACCAGGTTCCCAGGTTGTCGACGGCTACTACCAGCGCGCAGACCTCTG

[0230] GAAGGACCTTGAGGCCATGGGCTTCTACCTCTCCGGCTTCGGCTGCACCACCTGTATTG

[0231] GTAACTCCGGCCCACTGCCAGAGGAAATCTCCGCTGCGATCAACGAGCACGACCTGAC

[0232] CGCAACCGCAGTTTTGTCCGGTAACCGTAACTTCGAGGGACGTATCTCCCCTGACGTTA

[0233] AGATGAACTACCTGGCATCCCCAATCATGGTCATTGCTTACGCAATCGCTGGCACCATGG

[0234] ACTTCGACTTCGAGAACGAAGCTCTTGGACAGGACCAGGACGGCAACGACGTCTTCCT

[0235] GAAGGACATCTGGCCTTCCACCGAGGAAATCGAAGACACCATCCAGCAGGCAATCTCC

[0236] CGTGAGCTTTACGAAGCTGACTACGCAGATGTCTTCAAGGGTGACAAGCAGTGGCAGG

[0237] AACTCGATGTTCCTACCGGTGACACCTTCGAGTGGGACGAGAACTCCACCTACATCCGC

[0238] AAGGCACCTTACTTCGACGGCATGCCTGTCGAGCCAGTGGCAGTCACCGACATCCAGGG

[0239] CGCACGCGTTCTGGCTAAGCTCGGCGACTCTGTCACCACCGACCACATCTCCCCTGCTT

[0240] CCTCCATTAAGCCAGGTACCCCTGCAGCTCAGTACTTGGATGAGCACGGTGTGGAACGC

[0241] CACGACTACAACTCCCTGGGTTCCAGGCGTGGTAACCACGAGGTCATGATGCGCGGCAC

[0242] CTTCGCCAACATCCGCCTCCAGAACCAGCTGGTTGACATCGCAGGTGGCTACACCCGCG

[0243] ACTTCACCCAGGAGGGTGCTCCACAGGCGTTCATCTACGACGCTTCCGTCAACTACAAG

[0244] GCTGCTGGCATTCCGCTGGTCGTCTTGGGCGGCAAGGAGTACGGCACCGGTTCTTCCCG

[0245] TGACTGGGCAGCTAAGGGCACTAACCTGCTCGGAATTCGCGCAGTTATCACCGAGTCCT

[0246] TCGAGCGTATTCACCGCTCCAACCTCATCGGTATGGGCGTTGTCCCACTGCAGTTCCCTG

[0247] CAGGCGAATCCCACGAGTCCCTGGGCCTTGACGGCACCGAGACCTTCGACATCACCGG

[0248] ACTGACCGCACTCAACGAGGGCGAGACTCCTAAGACTGTCAAGGTCACCGCAACCAAG

[0249] GAGAACGGCGACGTCGTCGAGTTCGACGCAGTTGTCCGCATCGACACCCCAGGTGAGG

[0250] CTGACTACTACCGCCACGGCGGCATCCTGCAGTACGTGCTGCGTCAGATGGCTGCTTCTTCTAAGTAA(SEQ ID NO:8)

[0251] Corynebacterium glutamicum pEC-XK99E plasmid sequence:

[0252] GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGG

[0253] CTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAA

[0254] GCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCC

[0255] ATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGC

[0256] GAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGC

[0257] CTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGG

[0258] AGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCA

[0259] TAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTT

[0260] TCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTA

[0261] ATTCCGCTAGATGACGTGCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGCGCTCG

[0262] CGGCTGGCTGCGGCACGACACGCGTCTGAGCAGTATTTTGCGCGCCGTCCTCGTGGGTC

[0263] AGGCCGGGGTGGGATCAGGCCACCGCAGTAGGCGCAGCTGATGCGATCCTCCACTACTG

[0264] CGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCGGCCAGCTCTTCAAGGTCGGCC

[0265] ACAAGCGTTTCTAGGTCGCTCGCGGCACTTGCCCAGTCGCGTGATGCTGGCGCGTCTGT

[0266] CGTATCGAGGGCGCGGAAAAATCCGATCACCGTTTTTAAATCGACGGCGGCATCGAGTG

[0267] CGTCGGACTCCAGCGCGACATCGGAGAGATCCACCGCTGATGCTTCAGGCCAGTTTTGG

[0268] TACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTTAAAAATTCTAGCCCC

[0269] AATTCTGATAATTTCTTCCGGCACTCCTGCGAAAACCTGCGAGACTTCTTGCCCAGAAA

[0270] AAACGCCAAGCGCAGCGGTTACCGCACTTTTTTTCCAGGTGATTTCACCCTGACCAGCG

[0271] AAGCGGCACTTTAGTGCATGAGGTGTGCCCCTGGTTTCCCCTCTTTGGAGGGTTCAACC

[0272] CAAAAAAGCACACAAGCAAAAATGAAAATCATCATGAGCAAGTTGGTGCGAAGCAGCA

[0273] ACGCGCTAGCTCCAAAAAGGTCTCCAGGATCTCGAGGAGATTTTTGAGGGGGAGGGAG

[0274] TCGAGGAAGAGCCAGAGCAGAAGGCGGGGAACCGTTCTCTGCCGACAGCGTGAGCCC

[0275] CCCTTAAAAATCAGGCCGGGGAGGAACCGGGGAGGGATCAGAGCTAGGAGCGAGACA

[0276] CCCTAAAGGGGGGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGT

[0277] GGATAGCGGAGGGTGAGGGCAAGTGAGAGCCAGAGCAAGGACGGGACCCCTAAAGGG

[0278] GGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGACGGCCGCG

[0279] TTTAGCTTCCATTCCAAGTGCCTTTCTGACTTGTTGGATGCGCCTTTCACTGACACCTAG

[0280] TTCGCCTGCAAGCTCACGAGTCGAGGGATCAGCAACCGATTGAGAACGGGCATCCAGG

[0281] ATCGCAGTTTTGACGCGAAGTTCGAGCAACTCGCCTGTCATTTCTCGGCGTTTGTTTGCT

[0282] TCCGCTAATCGCTGTCGCGTCTCCTGCGCATACTTACTTTCTGGGTCAGCCCATCTGCGT

[0283] GCATTCGATGTAGCTGCGCCCCGTCGCCCCATCGTCGCTAGAGCTTTCCGCCCTCGGCTG

[0284] CTCTGCGTTTCCACCCGACGAGCAGGGACGACTGGCTGGCCTTTAGCCACGTAGCCGCG

[0285] CACACGACGCGCCATCGTCAGGCGATCACGCATGGCGGGAAGATCCGGCTCCCGGCCG

[0286] TCTGCACCGACCGCCTGGGCAACGTTGTACGCCACTTCATACGCGTCGATGATCTTGGCA

[0287] TCTTTTAGGCGCTCACCAGCAGCTTTGAGCTGGTATCCCACGGTCAACGCGTGGCGAAA

[0288] CGCGGTCTCGTCGCGCGCTCGCTCTGGATTTGTCCAGAGCACTCGCACGCCGTCGATCA

[0289] GGTCGCCGGACGCGTCCAGGGCGCTCGGCAGGCTCGCGTCCAAAATCGCTAGCGCCTT

[0290] GGCTTCTGCGGTGGCGCGTTGTGCCGCTTCAATGCGGGCGCGTCCGCTGGAAAAGTCCT

[0291] GCTCAATGTACTTTTTCGGCTTCTGTGATCCGGTCATCGTTCGAGCAATCTCCATTAGGTC

[0292] GGCCAGCCGATCCACACGATCATGCTGGCAGTGCCATTTATAGGCTGTCGGATCGTCTGA

[0293] GACGTGCAGCGGCCACCGGCTCAGCCTATGCGAAAAAGCCTGGTCAGCGCCGAAAACA

[0294] CGAGTCATTTCTTCCGTCGTTGCAGCCAGCAGGCGCATATTTGGGCTGGTTTTACCTGCT

[0295] GCGGCATACACCGGGTCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAGCGGATTCAC

[0296] GCCGATCCAAGCCGGCGCTTTTTCTAGGCGTGCCCATTTCTCTAAAATCGCGTAGACCTG

[0297] CGGGTTTACGTGCTCAATCTTCCCGCCGGCCTGGTGGCTGGGCACATCGATGTCAAGCA

[0298] CGATCACCGCGGCATGTTGCGCGTGCGTCAGCGCAACGTACTGGCACCGCGTCAGCGCT

[0299] TTTGAGCCAGCCCGGTAGAGCTTTGGTTGGGTTTCGCCGGTATCCGGGTTTTTAATCCAG

[0300] GCGCTCGCGAAATCTCTTGTCTTGCTGCCCTGGAAGCTTTCGCGTCCCAGGTGAGCGAG

[0301] CAGTTCGCGGCGATCTTCTGCCGTCCAGCCGCGTGAGCCGCAGCGCATAGCTTCGGGGT

[0302] GGGTGTCGAACAGATCGGCGGACAATTTCCACGCGCTAGCTGTGACTGTGTCCTGCGGA

[0303] TCGGCTAGAGTCATGTCTTGAGTGCTTTCTCCCAGCTGATGACTGGGGGTTAGCCGACG

[0304] CCCTGTGAGTTCCCGCTCACGGGGCGTTCAACTTTTTCAGGTATTTGTGCAGCTTATCGT

[0305] GTTTTCTTCGTAAATGAACGCTTAACTACCTTGTTAAACGTGGCAAATAGGCAGGATTGA

[0306] TGGGGATCTAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAG

[0307] CGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCT

[0308] ACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCA

[0309] GTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAA

[0310] TTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGG

[0311] CTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGA

[0312] TGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTG

[0313] GGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCG

[0314] CCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC

[0315] GGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG

[0316] GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTA

[0317] TTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGT

[0318] ATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATT

[0319] CGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTT

[0320] GTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCG

[0321] CCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGC

[0322] CTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG

[0323] GCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAG

[0324] AGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT

[0325] CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTT

[0326] CGCGGAATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC

[0327] CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTT

[0328] GCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA

[0329] ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA

[0330] GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

[0331] CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG

[0332] GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT

[0333] GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

[0334] GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC

[0335] GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT

[0336] CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGT

[0337] CAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGC

[0338] CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC

[0339] CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG

[0340] CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATC

[0341] TGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA

[0342] GTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACAC

[0343] CCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAG

[0344] ACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA

[0345] AACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTACGTT

[0346] GACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAG

[0347] TCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCG

[0348] GTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAA

[0349] ACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGG

[0350] CACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCC

[0351] CTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGC

[0352] CAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTG

[0353] CACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAG

[0354] GATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTG

[0355] ACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTG

[0356] GAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTC

[0357] TGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCA

[0358] GCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATG

[0359] CAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGC

[0360] GCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGG

[0361] TAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCACCATCA

[0362] AACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG

[0363] GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCA

[0364] CCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGC

[0365] TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA

[0366] GTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTT

[0367] CTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCA

[0368] TAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATA

[0369] ACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATG (SEQ ID NO: 9) trc promoter sequence (arrow indicates the transcription start direction):

[0370] TTGACAATTAATCATCCGGCTCGTATAATG → (SEQ ID NO: 10)

[0371] rrnB T1 terminator sequence (arrow indicates the transcription termination direction):

[0372] →

[0373] CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGA GTAGGACAAAT(SEQ ID NO:11)

[0374] LacI protein expression cassette (including promoter, CDS region, terminator):

[0375] GACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAG

[0376] TCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCG

[0377] GTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAA

[0378] ACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGG

[0379] CACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCC

[0380] CTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGC

[0381] CAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTG

[0382] CACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAG

[0383] GATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTG

[0384] ACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTG

[0385] GAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTC

[0386] TGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCA

[0387] GCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATG

[0388] CAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGC

[0389] GCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGG

[0390] TAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCACCATCA

[0391] AACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG

[0392] GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCA

[0393] CCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGC

[0394] TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA

[0395] GTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTT

[0396] CTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGG(SEQ ID NO: 12)

[0397] LacI protein binding site sequence:

[0398] GGAATTGTGAGCGGATAACAATTCC(SEQ ID NO: 13)

[0399] Saccharomyces cerevisiae related sequence:

[0400] Full sequence of the CDS region of the Saccharomyces cerevisiae ACO1 gene:

[0401] ATGCTGTCTGCACGTTCTGCCATCAAGAGACCCATTGTTCGTGGTCTTGCGACAGTCTCC

[0402] AACTTGACTAGAGATTCAAAAGTCAACCAAAACTTATTAGAAGATCATTCTTTTATTAAC

[0403] TACAAGCAGAATGTGGAAACGCTGGATATCGTAAGAAAAAGATTAAACAGGCCATTTAC

[0404] CTACGCGGAAAAGATTTTGTACGGTCACTTGGATGACCCTCATGGTCAAGATATTCAGAG

[0405] AGGTGTTTCATACCTAAAATTAAGACCAGATCGTGTTGCCTGTCAAGATGCTACTGCTCA

[0406] AATGGCTATTTTACAATTTATGTCCGCTGGTTTACCACAGGTTGCTAAGCCAGTCACTGTC

[0407] CACTGTGACCATTTGATTCAAGCACAAGTTGGTGGTGAAAAAGATTTGAAGAGAGCTAT

[0408] AGATCTAAACAAGGAAGTTTATGATTTCTTGGCCTCTGCCACTGCGAAATATAACATGGG

[0409] TTTCTGGAAGCCAGGTTCCGGTATCATTCACCAAATTGTTCTGGAAAACTACGCTTTCCC

[0410] AGGTGCTTTGATCATTGGTACTGACTCCCATACACCAAATGCTGGTGGTTTAGGTCAATT

[0411] GGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATGGCAGGTCGTCCATGGGAATT

[0412] GAAGGCTCCAAAGATCTTAGGTGTTAAGTTGACTGGTAAGATGAACGGTTGGACTTCTC

[0413] CAAAGGATATTATTTTGAAATTGGCTGGTATCACAACTGTCAAAGGTGGTACTGGTAAAA

[0414] TTGTTGAATATTTCGGTGATGGTGTTGACACCTTCTCCGCTACTGGTATGGGTACCATTTG

[0415] TAATATGGGTGCTGAAATCGGTGCTACCACATCTGTTTTCCCATTCAACAAATCTATGATT

[0416] GAATATTTGGAAGCAACTGGTCGTGGTAAGATCGCTGACTTTGCTAAATTATACCACAAG

[0417] GACCTATTATCTGCTGATAAGGATGCTGAATACGATGAGGTCGTCGAAATTGACTTGAAC

[0418] ACTCTGGAACCATACATCAATGGGCCATTTACCCCCGATTTGGCTACTCCAGTTTCTAAG

[0419] ATGAAGGAAGTTGCTGTTGCTAATAACTGGCCATTGGATGTCAGAGTCGGTTTGATCGGT

[0420] TCTTGTACCAATTCCTCTTATGAAGATATGTCTCGTTCAGCATCCATTGTCAAGGATGCTG

[0421] CTGCTCATGGTTTGAAATCCAAGACCATTTTCACTGTTACTCCAGGTTCTGAACAAATCA

[0422] GAGCCACTATTGAACGTGATGGCCAATTAGAAACCTTCAAAGAATTTGGTGGTATCGTTT

[0423] TGGCAAACGCCTGTGGCCCATGTATTGGTCAATGGGATCGTAGAGATATCAAGAAAGGT

[0424] GACAAGAATACTATCGTTTCCTCTTACAACAGAAATTTCACTTCTAGAAATGATGGTAAC

[0425] CCACAAACTCATGCTTTTGTTGCATCTCCAGAATTAGTAACTGCGTTCGCCATTGCGGGT

[0426] GATTTGAGATTCAACCCTCTAACAGACAAATTAAAGGACAAGGATGGTAATGAGTTCAT

[0427] GTTGAAACCACCACATGGTGATGGTTTGCCTCAAAGAGGTTATGATGCTGGTGAGAACA

[0428] CTTACCAAGCTCCACCTGCAGACCGTAGCACCGTTGAAGTTAAAGTTTCTCCAACTTCA

[0429] GACCGTCTACAACTGTTGAAACCATTCAAACCTTGGGATGGTAAGGATGCTAAAGACAT

[0430] GCCAATCTTGATTAAGGCCGTCGGTAAGACAACTACTGATCATATTTCTATGGCTGGTCCA

[0431] TGGTTGAAATACAGAGGTCATTTAGAAAACATTTCTAATAACTATATGATTGGTGCTATTA

[0432] ATGCTGAAAACAAGAAGGCTAACTGTGTTAAAAATGTATATACTGGTGAATACAAAGGT

[0433] GTTCCAGACACTGCTAGAGATTACAGAGACCAAGGTATCAAGTGGGTTGTTATTGGTGA

[0434] TGAAAACTTTGGTGAAGGTTCCTCTCGTGAACACGCTGCTTTGGAACCAAGATTCTTGG

[0435] GCGGTTTCGCTATCATCACAAAGTCTTTCGCTCGTATCCATGAAACTAACTTGAAAAAAC

[0436] AAGGTCTATTGCCATTGAACTTCAAGAACCCAGCTGACTATGACAAGATCAACCCTGAT

[0437] GACAGAATCGATATTCTGGGTCTAGCTGAATTGGCTCCAGGTAAGCCTGTAACAATGAG

[0438] AGTTCATCCAAAGAATGGTAAGCCATGGGATGCTGTGTTGACCCATACTTTCAACGATGA

[0439] GCAAATTGAATGGTTCAAATATGGTTCTGCCTTAAATAAAATTAAGGCCGATGAGAAGAAATAA(SEQ ID NO: 14)

[0440] Saccharomyces cerevisiae 2μm shuttle plasmid sequence:

[0441] CAACTTTGTATAGAAAAGTTGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGA

[0442] AGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATG

[0443] TGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATG

[0444] AAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATT

[0445] AACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAG

[0446] CGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTT

[0447] AACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCA

[0448] ACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCAAGTTTGT

[0449] ACAAAAAAGCAGGCTGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTG

[0450] GTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCG

[0451] GCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC

[0452] CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGT

[0453] GCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCC

[0454] GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCC

[0455] GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAT

[0456] CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGC

[0457] CACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT

[0458] CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

[0459] CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGC

[0460] CCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC

[0461] GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAACCCAGCTTTCTTGTAC

[0462] AAAGTGGTAGCGGCCGCTCGAGCATGCATCTAGAGGGCCGCATCATGTAATTAGTTATGT

[0463] CACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGA

[0464] CAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATT

[0465] TATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGA

[0466] AAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCC

[0467] TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

[0468] GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC

[0469] TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA

[0470] TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT

[0471] TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG

[0472] TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT

[0473] GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG

[0474] AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG

[0475] CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG

[0476] GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC

[0477] ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG

[0478] GTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC

[0479] AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA

[0480] GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA

[0481] GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGG

[0482] GATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

[0483] AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA

[0484] TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC

[0485] CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT

[0486] ACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAA

[0487] GGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT

[0488] GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT

[0489] GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC

[0490] CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT

[0491] CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGC

[0492] AGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG

[0493] TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC

[0494] GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA

[0495] ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT

[0496] AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT

[0497] GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAAT

[0498] GTTGAATACTCATACTCTTCCTTTTTCAATGGGTAATAACTGATATAATTAAATTGAAGCTC

[0499] TAATTTGTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTC

[0500] AAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACG

[0501] AGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGC

[0502] TATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGC

[0503] GGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCT

[0504] CGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGA

[0505] TGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGC

[0506] TCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAG

[0507] CCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACA

[0508] GGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACA

[0509] ACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTG

[0510] CCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGG

[0511] GCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGT

[0512] GCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCC

[0513] ATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTT

[0514] AAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAA

[0515] GTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTC

[0516] AAGGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAAAGCTAGCT

[0517] TATCGATGATAAGCTGTCAAACATGAGAATTAATTCCACGGACTATAGACTATACCTAGTA

[0518] TACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTAC

[0519] CACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCT

[0520] TCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTT

[0521] CTACAATGGCTGCCATCATTATTATCCGATGTGACGCTGCAGCTTCTCAATGATATTCGAA

[0522] TACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTA

[0523] CTTGTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATA

[0524] GTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATT

[0525] TCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCG

[0526] GTTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTG

[0527] TGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAAT

[0528] CTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAG

[0529] AATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACA

[0530] AAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCA

[0531] ACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCT

[0532] AACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGAT

[0533] AACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCT

[0534] CTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGG

[0535] GTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCAT

[0536] ACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACG

[0537] GTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTC

[0538] GATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATA

[0539] AACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATG

[0540] GGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGT

[0541] TTGTGGAAGCGGTATTCGCAATGGGAAGCTCCACCCCGGTTGATAATCAGAAAAGCCCC

[0542] AAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAGCGTTAATATTTTGTTAAAAT

[0543] TCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAAT

[0544] CCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACA

[0545] AGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA

[0546] GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCC

[0547] GTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAA

[0548] GCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGG

[0549] CGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT(SEQ ID NO:15)

[0550] Neomycin sulfate resistance gene sequence of Saccharomyces cerevisiae (including promoter, expression cassette,Terminator): GTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTG(SEQ ID NO: 16),

[0551] Shuttle vector Escherichia coli replicon sequence:

[0552] TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT

[0553] GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG

[0554] CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA

[0555] AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC

[0556] TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG

[0557] TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA

[0558] CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG

[0559] TGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCA

[0560] GTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAG

[0561] CGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA(SEQ ID NO: 17)

[0562]

[0563] Constitutive promoter TEF1 sequence of Saccharomyces cerevisiae:

[0564] GATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTC

[0565] TCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTCC

[0566] CCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAA

[0567] AGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTT

[0568] CTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTA

[0569] AGTTAATAAATGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG(SEQ ID NO: 19) LacI protein expression cassette for Saccharomyces cerevisiae (including promoter, expression cassette, terminator):

[0570] GATCCTTTTTTGTAGAAATGTCTTGGTGTCCTCGTCCAATCAGGTAGCCATCTCTGAAATA

[0571] TCTGGCTCCGTTGCAACTCCGAACGACCTGCTGGCAACGTAAAATTCTCCGGGGTAAAA

[0572] CTTAAATGTGGAGTAATGGAACCAGAAACGTCTCTTCCCTTCTCTCTCCTTCCACCGCCC

[0573] GTTACCGTCCCTAGGAAATTTTACTCTGCTGGAGAGCTTCTTCTACGGCCCCCTTGCAGC

[0574] AATGCTCTTCCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGCAG

[0575] CCCAGGGATGGAAAAGTCCCGGCCGTCGCTGGCAATAATAGCGGGCGGACGCATGTCAT

[0576] GAGATTATTGGAAACCACCAGAATCGAATATAAAAGGCGAACACCTTTCCCAATTTTGGT

[0577] TTCTCCTGACCCAAAGACTTTAAATTTAATTTATTTGTCCCTATTTCAATCAATTGAACAA

[0578] CTATCAAATGAAACCAGTTACTTTGTATGATGTTGCTGAATATGCTGGTGTTTCTTATCAA

[0579] ACTGTTTCTAGAGTTGTTAATCAAGCTTCTCATGTTTCTGCTAAAACTAGAGAAAAAGTT

[0580] GAAGCTGCTATGGCTGAATTGAATTATATTCCAAATAGAGTTGCTCAACAATTGGCTGGT

[0581] AAACAATCTTTGTTGATTGGTGTTGCTACTTCTTCTTTGGCTTTGCATGCTCCATCTCAAA

[0582] TTGTTGCTGCTATTAAATCTAGAGCTGATCAATTGGGTGCTTCTGTTGTTGTTTCTATGGT

[0583] TGAAAGATCTGGTGTTGAAGCTTGTAAAGCTGCTGTTCATAATTTGTTGGCTCAAAGAGT

[0584] TTCTGGTTTGATTATTAATTATCCATTGGATGATCAAGATGCTATTGCTGTTGAAGCTGCTT

[0585] GTACTAATGTTCCAGCTTTGTTTTTGGATGTTTCTGATCAAACTCCAATTAATTCTATTATT

[0586] TTTTCTCATGAAGATGGTACTAGATTGGGTGTTGAACATTTGGTTGCTTTGGGTCATCAA

[0587] CAAATTGCTTTGTTGGCTGGTCCATTGTCTTCTGTTTCTGCTAGATTGAGATTGGCTGGTT

[0588] GGCATAAATATTTGACTAGAAATCAAATTCAACCAATTGCTGAAAGAGAAGGTGATTGGT

[0589] CTGCTATGTCTGGTTTTCAACAAACTATGCAAATGTTGAATGAAGGTATTGTTCCAACTG

[0590] CTATGTTGGTTGCTAATGATCAAATGGCTTTGGGTGCTATGAGAGCTATTACTGAATCTGG

[0591] TTTGAGAGTTGGTGCTGATATTTCTGTTGTTGGTTATGATGATACTGAAGATTCTTCTTGT

[0592] TATATTCCACCATCTACTACTATTAAACAAGATTTTAGATTGTTGGGTCAAACTTCTGTTG

[0593] ATAGATTGTTGCAATTGTCTCAAGGTCAAGCTGTTAAAGGTAATCAATTGTTGCCAGTTT

[0594] CTTTGGTTAAAAGAAAAACTACTTTGGCTCCAAATACTCAAACTGCTTCTCCAAGAGCT

[0595] TTGGCTGATTCTTTGATGCAATTGGCTAGACAAGTTTCTAGATTGGAATCTGGTCAATGA

[0596] CTGGGATTACACATGGCATGGATGAACTATACAAATAATATATATATATATATATATATATATA

[0597] GTATCAAAAAAGGGGGGAAAGGGCCCCCCTTTCAAAGATCCACTAGTTCTAGAGCGGCCGCCACCGCGTGGAGCTCCAGCTTTTGTT(SEQ ID NO:20)

[0598] Yeast terminator CYC1 sequence:

[0599] ATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCG

[0600] AAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTT

[0601] AGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACG

[0602] CATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCT(SEQ ID NO:21)

[0603] The beneficial effects of the present invention are as follows:

[0604] The present invention designs an antisense RNA-based and modular plasmid elimination technology. The components required for constructing the plasmid elimination module are simple and easy to obtain, the plasmid module elimination efficiency is high, the elimination target can be freely set according to needs, and selective or synchronous elimination of multiple plasmids can be achieved. Moreover, the present invention has a wide application range, and this technology can be applied in both eukaryotes and prokaryotes. Antisense RNAs related to genes in the central metabolic pathway can all be used for plasmid elimination, and antisense RNAs of genes related to the metabolism of amino acids, nucleic acids, lipids, transcription and translation, DNA replication, cell wall, cell membrane, endoplasmic reticulum, etc. outside the central metabolic pathway can also be applied to this technology. If the elimination effect is not good, strategies such as increasing the types of antisense RNAs and extending the length of antisense RNAs can be used to strengthen the elimination effect. Brief Description of the Drawings

[0605] Figure 1 It is the interference result diagram of Example 5.

[0606] Figure 2 It is the interference result diagram of Example 6.

[0607] Figure 3 It is the interference result diagram of Example 7.

[0608] Figure 4 It is the interference result diagram of Example 8.

[0609] Figure 5 It is the schematic diagram of the principle of the present invention. Detailed implementation manners

[0610] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0611] The principle of the antisense RNA elimination plasmid is as follows: We place the antisense RNA that can specifically bind to the mRNAs of key metabolic genes such as genes related to the TCA cycle, protein synthesis, DNA / RNA synthesis, amino acid synthesis, ribose synthesis, ribosome synthesis, and ribosome assembly genes downstream of the repressor protein binding site, and control the transcription of the antisense RNA of the key metabolic genes by adding / removing the repressor protein. 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, making the growth rate of the cells expressing the antisense RNA significantly lower than that of the cells not expressing the antisense RNA. Since the plasmid has the phenomenon of spontaneous loss, the plasmid-free cells can be enriched by subculture to achieve plasmid elimination. (The principle is as Figure 5 shown)

[0612] The repressor protein can controllably inhibit the expression of the antisense RNA of the key metabolic gene on the plasmid to be eliminated, thereby achieving controllable cell growth inhibition, controllable enrichment of plasmid-free cells, and plasmid elimination.

[0613] Instrumentation:

[0614] 1. PCR instrument

[0615] 2. Electrophoresis instrument

[0616] 3. Electrophoresis tank

[0617] 4. Ultra-low temperature refrigerator

[0618] 5. Biochemical incubator

[0619] 6. Autoclave

[0620] 7. Water bath

[0621] 8. Medical freezer

[0622] 9. Tabletop centrifuge

[0623] 10. Pipette

[0624] 11. Oven

[0625] 12. Electroporator and its supporting 1mm electroporation cuvette Reagents:

[0626]

[0627]

[0628] Biological materials:

[0629] E. coli related plasmids and primers:

[0630] Primers:

[0631] EP001:

[0632] CTCGCTCTCATTGTCATAGTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGG(SEQ IDNO: 22)

[0633] EP002:

[0634] GACAATGAGAGCGAGGAGAAGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCG(SEQ IDNO: 23)

[0635] EP003: CATCGGTGATGTCGGCGATATAGG(SEQ ID NO: 24)

[0636] EP004: GCTAATCGATGATAAGCTGTCAAATTTCGCGGGATCGAGATCTCGATCCTC(SEQ ID NO: 25)

[0637] EP005: TTGACAGCTTATCATCGATTAGCTTTAATGGAATTGTGAGCGGATAACAATTCC(SEQ IDNO: 26)

[0638] EP006:

[0639] GAATACCGTAAGCACGTAGCTGAGCGTGCCGCTGGGAATTGTTATCCGCTCACAATTCC(SEQ IDNO: 27)

[0640] EP007:

[0641] CTACGTGCTTACGGTATTCTTCTAGCACGACGGTTCTCCTCGCTCTCATTGTCATAGTC(SEQ ID NO: 28)

[0642] Corynebacterium glutamicum-related plasmids and primers:

[0643] Primers:

[0644] GP001:

[0645] CCTTCAATGCTAAGAGCACCCTTGAAGTTGGCGACAAGTCCTTGTTATCCGCTCACAATT CC(SEQ ID NO: 29)

[0646] GP002:

[0647] GGGTGCTCTTAGCATTGAAGGAGCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGG(SEQ ID NO: 30)

[0648] GP003: GCTGTGCAGGTCGTAAATCACTGC(SEQ ID NO: 31)

[0649] Saccharomyces cerevisiae-related plasmids and primers:

[0650] Primers:

[0651] SP001: CGTATAACGTTACTGGTTTCACGGTGGCAGCCTGCTTTTTTGTACAAACTTGC(SEQ ID NO: 32)

[0652] SP002: GTGAAACCAGTAACGTTATACGATGTCGC(SEQ ID NO: 33)

[0653] SP003: GTTCACTGCCCGCTTTCCAGTCG(SEQ ID NO: 34)

[0654] SP004: GGAAAGCGGGCAGTGAACCCAGCTTTCTTGTACAAAGTGGTAGC(SEQ ID NO: 35) SP005: GAAGAAAGCGAAAGGAGCGGAGCTTGCAAATTAAAGCCTTCGAGC(SEQ ID NO: 36)

[0655] SP006: CCGCTCCTTTCGCTTTCTTCCCTTCC (SEQ ID NO: 37)

[0656] SP007: GCGGCGCATTAAGCGAGTTTACCACACACCATAGCTTCAAAATGTTTC (SEQ ID NO: 38)

[0657] SP008: TAAACTCGCTTAATGCGCCGCTACAGG (SEQ ID NO: 39)

[0658] SP009:

[0659] CAAGACCACGAACAATGGGTCTCTTGATGGCAGAACGTGCATCATGTAATTAGTTATGTC ACGC (SEQID NO: 40)

[0660] SP010: CCATTGTTCGTGGTCTTGCGACAGTCTCCAACTTGACTAGAGTTGTGAGCGGATAACAACTTAGATTAGATTGCTATGCTTTC (SEQ ID NO: 41)

[0661] SP011: GATTTAGAGCTTGACGGGGAAAGCC (SEQ ID NO: 42)

[0662] SP012: CTGATTCTGTGGATAACCGTATTACC (SEQ ID NO: 43)

[0663] Example 1 Construction of E. coli T7 promoter + LacI repressor plasmid elimination module

[0664] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0665] 1. Design primers EP001 and EP002 and amplify the plasmid template pET28a plasmid system according to the following system and PCR program:

[0666]

[0667] PCR program:

[0668]

[0669] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR product EP001-EP002 according to the purification kit instructions.

[0670] 3. Connect and purify the product EP001 - EP002 according to the operation procedure shown in the instruction manual of the ready - to - use seamless cloning kit.

[0671] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[0672] 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.

[0673] 2) Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[0674] 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.

[0675] 4) Take an appropriate volume of the recovered solution and spread it evenly on an LB solid antibiotic medium containing 50 μg / mL kanamycin, and incubate it upside - down in a 37 °C incubator overnight.

[0676] (2) Sequencing verification experiment for the construction result:

[0677] Take the single colonies obtained from the spread plate and send them for sequencing verification of the construction result using the EP003 primer.

[0678] Plasmid construction result:

[0679] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA

[0680] CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCT

[0681] ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT

[0682] TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCAC

[0683] TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

[0684] TATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA

[0685] TTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA

[0686] ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC

[0687] GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA

[0688] AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC

[0689] AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAA

[0690] CCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

[0691] CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAA

[0692] TATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGC

[0693] AGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG

[0694] GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT

[0695] ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGAT

[0696] TGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC

[0697] ATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA

[0698] CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAA

[0699] CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0700] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

[0701] CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC

[0702] AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

[0703] AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

[0704] GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

[0705] GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

[0706] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

[0707] AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0708] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

[0709] CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA

[0710] CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

[0711] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

[0712] ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0713] GAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATG

[0714] GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTAT

[0715] CGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC

[0716] TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG

[0717] CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA

[0718] GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCT

[0719] CGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGG

[0720] CGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG

[0721] TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT

[0722] GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0723] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCA

[0724] CAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA

[0725] CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTC

[0726] AGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCAT

[0727] TCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCAC

[0728] GATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAAC

[0729] GTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATAC

[0730] CGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATG

[0731] ACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAG

[0732] TGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT

[0733] CTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCG

[0734] TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0735] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA

[0736] CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG

[0737] CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACG

[0738] GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACC

[0739] AACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG

[0740] CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0741] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTG

[0742] AGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC

[0743] TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTAC

[0744] CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAAT

[0745] AACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0746] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC

[0747] AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0748] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG

[0749] TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA

[0750] ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG

[0751] CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGAC

[0752] ATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT

[0753] CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0754] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG

[0755] CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGG

[0756] GCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGA

[0757] TCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGT

[0758] GATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAAT

[0759] ACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT

[0760] TAACTTTAAGAAGGAGATATACCATGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC

[0761] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT. (SEQ ID NO: 44)

[0762] Among them, the antisense RNA sequence is ACTATGACAATGAGAGCGAGGAGAA. (SEQ ID NO: 45)

[0763] (3) Plasmid extraction experiment:

[0764] 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 Kit.

[0765] Example 2 Construction of Escherichia coli tet promoter + LacI repressor plasmid elimination module

[0766] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0767] 1. Design primers EP004 and EP005, and amplify the plasmid obtained from the construction of the "T7 promoter + LacI repressor plasmid elimination module" according to the following system and PCR program.

[0768] System:

[0769]

[0770] PCR program:

[0771]

[0772] 2. Use the ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR product EP004-EP005 according to the instructions of the purification kit.

[0773] 3. Connect the purified product EP004-EP005 according to the operation procedure shown in the instructions of the ready-to-use seamless cloning kit.

[0774] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[0775] 1) Take 100 μL of competent cells melted on ice, add the target DNA (plasmid or ligation product), and gently mix (pipette gently or flick the tube wall several times), then let it stand on ice for 5 min.

[0776] 2) Heat shock in a 42 °C water bath for 60 s, then 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).

[0777] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and then incubate at 37 °C and 200 rpm for 20 min (the transformation efficiency increases by 3 - 5 times for every 5 min increase in the recovery time).

[0778] 4) Take an appropriate volume of the recovered liquid and spread it evenly on an LB solid medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator overnight.

[0779] (2) Plasmid extraction experiment:

[0780] Select available single colonies on the transformation plate in (1) and 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 - kit.

[0781] (3) Second - round high - fidelity enzyme amplification and plasmid construction experiment:

[0782] 1. Design primers EP006 and EP007 and amplify the plasmid obtained in (2) according to the following system and PCR program

[0783] System:

[0784]

[0785]

[0786] PCR program:

[0787]

[0788] 2. Use a ready - to - use seamless cloning kit to assemble the SanPrep column - type PCR product purification kit, and purify the PCR product EP006 - EP007 according to the instructions of the purification kit.

[0789] 3. Connect the purified product EP006 - EP007 according to the operation procedure shown in the instructions of the ready - to - use seamless cloning kit.

[0790] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[0791] 1) Take 100 μL of competent cells melted on ice, add the ligation product in “3”, and gently mix (pipette gently or flick the tube wall several times), then let it stand on ice for 5 min.

[0792] 2) Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[0793] 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.

[0794] 4) Take an appropriate volume of the recovered liquid 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.

[0795] (4) Sequencing verification experiment for the construction result:

[0796] Take the single colonies obtained from the spread plate and send them for sequencing verification of the construction result using the EP003 primer.

[0797] Plasmid construction result:

[0798] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA

[0799] CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCT

[0800] ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT

[0801] TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCAC

[0802] TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

[0803] TATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA

[0804] TTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA

[0805] ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC

[0806] GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA

[0807] AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC

[0808] AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAA

[0809] CCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

[0810] CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAA

[0811] TATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGC

[0812] AGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG

[0813] GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT

[0814] ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGAT

[0815] TGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC

[0816] ATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA

[0817] CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAA

[0818] CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0819] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

[0820] CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC

[0821] AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

[0822] AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

[0823] GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

[0824] GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

[0825] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

[0826] AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0827] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

[0828] CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA

[0829] CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

[0830] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

[0831] ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0832] GAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATG

[0833] GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTAT

[0834] CGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC

[0835] TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG

[0836] CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA

[0837] GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCT

[0838] CGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGG

[0839] CGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG

[0840] TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT

[0841] GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0842] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCA

[0843] CAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA

[0844] CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTC

[0845] AGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCAT

[0846] TCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCAC

[0847] GATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAAC

[0848] GTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATAC

[0849] CGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATG

[0850] ACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAG

[0851] TGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT

[0852] CTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCG

[0853] TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0854] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA

[0855] CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG

[0856] CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACG

[0857] GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACC

[0858] AACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG

[0859] CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0860] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTG

[0861] AGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC

[0862] TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTAC

[0863] CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAAT

[0864] AACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0865] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC

[0866] AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0867] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG

[0868] TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA

[0869] ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG

[0870] CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGAC

[0871] ATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT

[0872] CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0873] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG

[0874] CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGG

[0875] GCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGA

[0876] TCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGT

[0877] GATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTTGA

[0878] CAGCTTATCATCGATTAGCTTTAATGGAATTGTGAGCGGATAACAATTCCCAGCGGCACG

[0879] CTCAGCTACGTGCTTACGGTATTCTTCTAGCACGACGGTTCTCCTCGCTCTCATTGTCATA

[0880] GTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT。(SEQ ID NO:46)

[0881] Among them, the antisense RNA sequence is:

[0882] CAGCGGCACGCTCAGCTACGTGCTTACGGTATTCTTCTAGCACGACGGTTCTCCTCGCTCTCATTGTCATAG。(SEQ ID NO:47)

[0883] Example 3 Construction of Corynebacterium glutamicum trc promoter + LacI repressor protein plasmid elimination module

[0884] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0885] 1. Design primers GP001 and GP002 and amplify the plasmid template pEC-XK99E plasmid system according to the following system and PCR program:

[0886]

[0887]

[0888] PCR program:

[0889]

[0890] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products EP001-EP002 according to the purification kit instructions.

[0891] 3. Connect the purified products GP001-GP002 according to the operation process shown in the ready-to-use seamless cloning kit instructions.

[0892] 4. Transform according to the following steps 5αChemically Competent Cell and culture overnight:

[0893] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (gently pipette or flick the tube wall several times), and let stand on ice for 5 min.

[0894] 2) Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0895] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and resuscitate at 37 °C and 200 rpm for 40 min.

[0896] 4) Take an appropriate volume of the resuscitation solution and spread it evenly on an LB solid medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator overnight.

[0897] (2) Sequencing verification experiment of the construction results

[0898] Pick the single colonies obtained from the spread plates and use the GP003 primer for sequencing verification of the construction results.

[0899] Experimental results:

[0900] CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCCGCTAGATGACGTGCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCGTCTGAGCAGTATTTTGCGCGCCGTCCTCGTGGGTCAGGCCGGGGTGGGATCAGGCCACCGCAGTAGGCGCAGCTGATGCGATCCTCCACTACTGCGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCGGCCAGCTCTTCAAGGTCGGCCACAAGCGTTTCTAGGTCGCT

[0901] CGCGGCACTTGCCCAGTCGCGTGATGCTGGCGCGTCTGTCGTATCGAGGGCGCGGAAAA

[0902] ATCCGATCACCGTTTTTAAATCGACGGCGGCATCGAGTGCGTCGGACTCCAGCGCGACA

[0903] TCGGAGAGATCCACCGCTGATGCTTCAGGCCAGTTTTGGTACTTCGTCGTGAAGGTCAT

[0904] GACACCATTATAACGAACGTTCGTTAAAAATTCTAGCCCCAATTCTGATAATTTCTTCCGG

[0905] CACTCCTGCGAAAACCTGCGAGACTTCTTGCCCAGAAAAAACGCCAAGCGCAGCGGTT

[0906] ACCGCACTTTTTTTCCAGGTGATTTCACCCTGACCAGCGAAGCGGCACTTTAGTGCATG

[0907] AGGTGTGCCCCTGGTTTCCCCTCTTTGGAGGGTTCAACCCAAAAAAGCACACAAGCAA

[0908] AAATGAAAATCATCATGAGCAAGTTGGTGCGAAGCAGCAACGCGCTAGCTCCAAAAAG

[0909] GTCTCCAGGATCTCGAGGAGATTTTTGAGGGGGAGGGAGTCGAGGAAGAGCCAGAGCA

[0910] GAAGGCGGGGAACCGTTCTCTGCCGACAGCGTGAGCCCCCCTTAAAAATCAGGCCGGG

[0911] GAGGAACCGGGGAGGGATCAGAGCTAGGAGCGAGACACCCTAAAGGGGGGGAACCGT

[0912] TTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGATAGCGGAGGGTGAGGGC

[0913] AAGTGAGAGCCAGAGCAAGGACGGGACCCCTAAAGGGGGGAACCGTTTTCTGCTGAC

[0914] GGTGTTTCGTTTATTAGTTTTCAGCCCGTGGACGGCCGCGTTTAGCTTCCATTCCAAGTG

[0915] CCTTTCTGACTTGTTGGATGCGCCTTTCACTGACACCTAGTTCGCCTGCAAGCTCACGAG

[0916] TCGAGGGATCAGCAACCGATTGAGAACGGGCATCCAGGATCGCAGTTTTGACGCGAAG

[0917] TTCGAGCAACTCGCCTGTCATTTCTCGGCGTTTGTTTGCTTCCGCTAATCGCTGTCGCGT

[0918] CTCCTGCGCATACTTACTTTCTGGGTCAGCCCATCTGCGTGCATTCGATGTAGCTGCGCC

[0919] CCGTCGCCCCATCGTCGCTAGAGCTTTCCGCCCTCGGCTGCTCTGCGTTTCCACCCGACG

[0920] AGCAGGGACGACTGGCTGGCCTTTAGCCACGTAGCCGCGCACACGACGCGCCATCGTC

[0921] AGGCGATCACGCATGGCGGGAAGATCCGGCTCCCGGCCGTCTGCACCGACCGCCTGGG

[0922] CAACGTTGTACGCCACTTCATACGCGTCGATGATCTTGGCATCTTTTAGGCGCTCACCAG

[0923] CAGCTTTGAGCTGGTATCCCACGGTCAACGCGTGGCGAAACGCGGTCTCGTCGCGCGCT

[0924] CGCTCTGGATTTGTCCAGAGCACTCGCACGCCGTCGATCAGGTCGCCGGACGCGTCCAG

[0925] GGCGCTCGGCAGGCTCGCGTCCAAAATCGCTAGCGCCTTGGCTTCTGCGGTGGCGCGTT

[0926] GTGCCGCTTCAATGCGGGCGCGTCCGCTGGAAAAGTCCTGCTCAATGTACTTTTTCGGC

[0927] TTCTGTGATCCGGTCATCGTTCGAGCAATCTCCATTAGGTCGGCCAGCCGATCCACACGA

[0928] TCATGCTGGCAGTGCCATTTATAGGCTGTCGGATCGTCTGAGACGTGCAGCGGCCACCG

[0929] GCTCAGCCTATGCGAAAAAGCCTGGTCAGCGCCGAAAACACGAGTCATTTCTTCCGTCG

[0930] TTGCAGCCAGCAGGCGCATATTTGGGCTGGTTTTACCTGCTGCGGCATACACCGGGTCAA

[0931] TGAGCCAGATGAGCTGGCATTTCCCGCTCAGCGGATTCACGCCGATCCAAGCCGGCGCT

[0932] TTTTCTAGGCGTGCCCATTTCTCTAAAATCGCGTAGACCTGCGGGTTTACGTGCTCAATC

[0933] TTCCCGCCGGCCTGGTGGCTGGGCACATCGATGTCAAGCACGATCACCGCGGCATGTTG

[0934] CGCGTGCGTCAGCGCAACGTACTGGCACCGCGTCAGCGCTTTTGAGCCAGCCCGGTAG

[0935] AGCTTTGGTTGGGTTTCGCCGGTATCCGGGTTTTTAATCCAGGCGCTCGCGAAATCTCTT

[0936] GTCTTGCTGCCCTGGAAGCTTTCGCGTCCCAGGTGAGCGAGCAGTTCGCGGCGATCTTC

[0937] TGCCGTCCAGCCGCGTGAGCCGCAGCGCATAGCTTCGGGGTGGGTGTCGAACAGATCG

[0938] GCGGACAATTTCCACGCGCTAGCTGTGACTGTGTCCTGCGGATCGGCTAGAGTCATGTC

[0939] TTGAGTGCTTTCTCCCAGCTGATGACTGGGGGTTAGCCGACGCCCTGTGAGTTCCCGCT

[0940] CACGGGGCGTTCAACTTTTTCAGGTATTTGTGCAGCTTATCGTGTTTTCTTCGTAAATGA

[0941] ACGCTTAACTACCTTGTTAAACGTGGCAAATAGGCAGGATTGATGGGGATCTAGCTTCAC

[0942] GCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAG

[0943] CCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACA

[0944] AGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGAT

[0945] AGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCC

[0946] CTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGA

[0947] TCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATG

[0948] ATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGG

[0949] CTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAG

[0950] CGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTC

[0951] CAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTG

[0952] TGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGG

[0953] GCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGC

[0954] AATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAAC

[0955] ATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTG

[0956] GACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGA

[0957] TGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGG

[0958] TGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGC

[0959] TATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

[0960] GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC

[0961] GCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCGGAATCATGACCAAAAT

[0962] CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT

[0963] CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC

[0964] TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT

[0965] GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA

[0966] CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT

[0967] GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTAC

[0968] CGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA

[0969] GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

[0970] CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA

[0971] GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT

[0972] TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT

[0973] GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT

[0974] CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT

[0975] GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGA

[0976] AGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG

[0977] CATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTC

[0978] CGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGAC

[0979] GCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC

[0980] CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAG

[0981] ATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTACGTTGACACCATCGAATGGTGC

[0982] AAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAA

[0983] TGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGT

[0984] TTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAA

[0985] GCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCA

[0986] AACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA

[0987] ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGAT

[0988] GGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAA

[0989] CGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAA

[0990] GCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAAC

[0991] AGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTG

[0992] GGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCG

[0993] TCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGA

[0994] AGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCA

[0995] TCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCC

[0996] ATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACC

[0997] GAAGACAGCTCATGTTATATCCCGCCGTCAACCACCATCAAACAGGATTTTCGCCTGCTG

[0998] GGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCA

[0999] ATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAA

[1000] ACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG

[1001] ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCGCGAATTGATCTGG

[1002] TTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCG

[1003] GAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGC

[1004] GCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCT

[1005] GAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAGGACTTGTCGCCAACTTCAAGGGTGCTCTTAGCATTGAAGGAG(SEQ ID NO: 48) The antisense RNA sequence:

[1006] GGACTTGTCGCCAACTTCAAGGGTGCTCTTAGCATTGAAGGAG(SEQ ID NO: 49)

[1007] (3) Plasmid extraction experiment:

[1008] 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, and activate them overnight. Complete the plasmid extraction according to the instructions of the Tiangen Plasmid Mini Kit.

[1009] Example 4 Construction of the Constitutive Promoter + LacI Repressor Protein Plasmid Elimination Module in Saccharomyces cerevisiae

[1010] (1) High-fidelity Enzyme Amplification and Plasmid Construction Experiment:

[1011] 1. Use primers SP001 - SP004 / SP009 - SP010 to amplify the plasmid template Saccharomyces cerevisiae 2μ plasmid according to the following system and PCR program; use primers SP002 - SP003 to amplify the codon-optimized LacI protein expression cassette.

[1012] System:

[1013]

[1014] PCR Program:

[1015]

[1016]

[1017] 2. Use the ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products SP001 - SP004 / SP009 - SP010 / SP002 - SP003 according to the instructions of the purification kit.

[1018] 3. Connect the purified products SP001 - SP004 and SP002 - SP003 respectively according to the operation procedure shown in the instructions of the ready-to-use seamless cloning kit; self-ligate the purified product SP009 - SP010 with the ready-to-use seamless cloning kit.

[1019] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[1020] 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.

[1021] 2) Heat shock in a 42°C water bath for 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[1022] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and resuscitate at 37°C and 200 rpm for 40 min.

[1023] 4) Take an appropriate volume of the resuscitation solution and evenly coat it on the LB solid medium containing 50 μg / mL kanamycin, and incubate it overnight in an inverted position in a 37 °C incubator.

[1024] (2) Construction result sequencing verification experiment:

[1025] Take the single colonies obtained from the two coated plates and use the SP011 / SP012 primers to send them for sequencing and verification of the construction results respectively, and obtain the SP001 - SP004 / SP002 - SP003 - 2μ plasmid and the SP009 - SP010 - 2μ plasmid.

[1026] (3) Plasmid extraction experiment:

[1027] According to the sequencing alignment results in (2), select the available single colonies on the transformation plate in (1) and 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 miniprep kit.

[1028] (4) High - fidelity enzyme amplification and plasmid construction experiment:

[1029] 1. The primers SP006 - SP008 amplify the correctly sequenced SP001 - SP004 / SP002 - SP003 - 2μ plasmid according to the following system and PCR program; the primers SP005 - SP007 amplify the correctly sequenced SP009 - SP010 - 2μ plasmid

[1030] System:

[1031]

[1032]

[1033] PCR program:

[1034]

[1035] 2. Use the ready - to - use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products SP006 - SP008 / SP005 - SP007 according to the instructions of the purification kit.

[1036] 3. Connect the purified products SP006 - SP008 and SP005 - SP007 according to the operation process shown in the instructions of the ready - to - use seamless cloning kit.

[1037] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[1038] 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.

[1039] 2) Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[1040] 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.

[1041] 4) Take an appropriate volume of the recovered solution and evenly spread it on an LB solid medium containing 100 μg / mL AMP antibiotic, and incubate it upside down in a 37 °C incubator overnight.

[1042] (2) Sequencing verification experiment for the construction result:

[1043] Take the single colonies obtained from the spread plate and use SP011 / SP012 primers to send for sequencing to verify the construction result.

[1044] Plasmid construction result:

[1045] CAACTTTGTATAGAAAAGTTGCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGCAAGTTTGTACAAAAAAGCAGGCTGCCACCGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCA

[1046] GCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTA

[1047] CATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTG

[1048] CCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGC

[1049] GCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAG

[1050] CCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAAC

[1051] TATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCG

[1052] TTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACG

[1053] GTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTA

[1054] GCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTC

[1055] ACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCG

[1056] GTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTT

[1057] GCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGT

[1058] TGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCC

[1059] GCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCT

[1060] TGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTG

[1061] GTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG

[1062] CCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAACC

[1063] CAGCTTTCTTGTACAAAGTGGTAGCGGCCGCTCGAGCATGCATCTAGAGGGCCGCATCA

[1064] TGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAA

[1065] AGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGT

[1066] ATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCAT

[1067] GTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTT

[1068] GCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA

[1069] TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG

[1070] CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAA

[1071] CGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC

[1072] CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGAC

[1073] GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT

[1074] GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC

[1075] TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG

[1076] GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG

[1077] CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCC

[1078] ACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA

[1079] GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGC

[1080] GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA

[1081] AACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAA

[1082] AAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA

[1083] AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTT

[1084] TTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACA

[1085] GTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT

[1086] AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCC

[1087] CAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA

[1088] ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC

[1089] CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGC

[1090] AACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA

[1091] TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAA

[1092] AGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC

[1093] ACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT

[1094] TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAG

[1095] TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT

[1096] GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGA

[1097] GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCA

[1098] CCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA

[1099] GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATGGGTAATAACTGATAT

[1100] AATTAAATTGAAGCTCTAATTTGTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTT

[1101] TCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCG

[1102] ATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATC

[1103] ACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGA

[1104] TGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGG

[1105] GTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGC

[1106] TGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCAT

[1107] CCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCG

[1108] GATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAG

[1109] CAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTT

[1110] CCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCC

[1111] ACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTG

[1112] ACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAG

[1113] CCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGA

[1114] ACCTGCGTGCAATCCATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTT

[1115] CTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGT

[1116] ATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCG

[1117] AATCAAAAAAATTTCAAGGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGA

[1118] ATTGAAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCCACGGACTA

[1119] TAGACTATACCTAGTATACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCT

[1120] TTAACGAGGCCTTACCACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTG

[1121] ATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAA

[1122] TGCAAAAGGCACTTCTACAATGGCTGCCATCATTATTATCCGATGTGACGCTGCAGCTTC

[1123] TCAATGATATTCGAATACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACA

[1124] GATTTACGATCGTACTTGTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTC

[1125] CCTGAAACAGATAGTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAG

[1126] ACAATGTATGTATTTCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCA

[1127] CGTCGCATCCCCGGTTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTA

[1128] ACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTT

[1129] CAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATT

[1130] TTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCT

[1131] AATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAG

[1132] CGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGA

[1133] GCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATG

[1134] CAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGG

[1135] TGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAG

[1136] CGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGT

[1137] GGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAA

[1138] AATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTT

[1139] CGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAA

[1140] TACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGA

[1141] AAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTT

[1142] TTGAGCAATGTTTGTGGAAGCGGTATTCGCAATGGGAAGCTCCACCCCGGTTGATAATC

[1143] AGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAGCGTTAATA

[1144] TTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGA

[1145] AATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCC

[1146] AGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAA

[1147] ACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGG

[1148] TCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG

[1149] ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG

[1150] AGCTTGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTAT

[1151] AATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATA

[1152] ACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCT

[1153] AACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACA

[1154] TAACTAATTACATGATGCACGTTCTGCCATCAAGAGACCCATTGTTCGTGGTCTTGCGAC

[1155] AGTCTCCAACTTGACTAGAGTTGTGAGCGGATAACAACTTAGATTAGATTGCTATGCTTT

[1156] CTTTCTAATGAGCAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAA

[1157] ACTTGAGAAATTGAAGACCGTTTATTAACTTAAATATCAATGGGAGGTCATCGAAAGAG

[1158] AAAAAAATCAAAAAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCC

[1159] TTTTTCGACGAAGAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTA

[1160] CGGGTAATTAACGACACCCTAGAGGAAGAAAGAGGGGAAATTTAGTATGCTGTGCTTGG

[1161] GTGTTTTGAAGTGGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAA

[1162] GGAGTAGAAACATTTTGAAGCTATGGTGTGTGGTAAACTCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT(SEQ ID NO:50)

[1163] The antisense RNA sequence is as follows:

[1164] CTCTAGTCAAGTTGGAGACTGTCGCAAGACCACGAACAATGGGTCTCTTGATGGCA GAACGTGC(SEQID NO:51)

[1165] Example 5 Plasmid curing using the Escherichia coli T7 promoter + LacI repressor protein plasmid curing module

[1166] 1. Prepare competent cells from the Escherichia coli to be transformed according to the procedure described in the Super Competent Cell Preparation Kit.

[1167] 2. Take 100 μL of the competent cells melted on ice, add the plasmid described in Example 1, mix gently (pipette gently or flick the tube wall a few times), and let stand on ice for 5 min.

[1168] 3. Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let stand for 2 min (do not shake the sample during the standing on ice, otherwise the transformation efficiency will be reduced).

[1169] 4. Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube and mix well.

[1170] 5. Take an appropriate volume of the resuscitation solution and spread it evenly on an LB solid medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator overnight.

[1171] 6. Add the frozen bacteria / puncture bacteria of Escherichia coli constructed in "5" or Escherichia coli containing the construction module in "5" into LB liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, at 37°C, 220 rpm.

[1172] 7. Add IPTG inducer with a final concentration of 1 mM to the LB medium in "6".

[1173] 8. After overnight induction of Escherichia coli / Corynebacterium glutamicum (12 h), streak on a solid LB medium plate without antibiotics.

[1174] Pick the single colonies obtained in "8" and spot them on solid medium plates without antibiotics and with antibiotics respectively (each single colony needs to be spotted on two plates to ensure the distinction of phenotypic differences).

[1175] 9. The specific results are as Figure 1 shown. After plasmid curing, most of the bacteria lost their resistance to antibiotics and could no longer grow on the antibiotic-containing plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully cured. Inoculate the corresponding strains with successful curing to obtain strains with successfully cured plasmids.

[1176] Example 6 Plasmid curing using the Escherichia coli tet promoter + LacI repressor plasmid curing module

[1177] 1. Prepare the Escherichia coli to be transformed into competent cells according to the procedure described in the super-competent cell preparation kit.

[1178] 2. Take 100 μL of competent cells melted on ice, add the plasmid described in Example 2 of the technical solution, mix gently (gently pipette or flick the tube wall several times), and let it stand on ice for 5 min.

[1179] 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).

[1180] 4. Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube and mix well.

[1181] 5. Take an appropriate volume of the recovery solution and evenly spread it on a solid LB medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37°C incubator overnight.

[1182] 6. Add the frozen bacteria / puncture bacteria of Escherichia coli constructed in "5" or Escherichia coli containing the construction module in "5" into antibiotic-free LB liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, at 37°C, 220 rpm.

[1183] 7. Add IPTG inducer with a final concentration of 1 mM to the LB described in "6".

[1184] 8. After overnight induction of Escherichia coli / Corynebacterium glutamicum (12 h), streak on a solid LB medium plate without antibiotics.

[1185] 9. Take the single colonies obtained in "8." and spot plate them on solid medium plates without antibiotics and with antibiotics respectively (each single colony needs to be spot plated twice to ensure the distinction of phenotypic differences).

[1186] 10. The specific results are as Figure 2 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 strains with successful curing can obtain strains with successfully cured plasmids.

[1187] Example 7 Plasmid curing using the trc promoter + LacI repressor plasmid curing module of Corynebacterium glutamicum

[1188] 1. Overnight activate the Corynebacterium glutamicum ATCC13032 strain in 5 ml of brain heart infusion liquid medium.

[1189] 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 at 200 rpm and 30 °C until the OD600 reaches 0.9.

[1190] 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 ul / tube into sterile 1.5 mL centrifuge tubes, and store at -70 °C or use immediately.

[1191] 4. During electroporation, thaw the aliquoted competent cells on ice and add the plasmid obtained in Example 3.

[1192] 5. Transfer the mixture to a cold electroporation cuvette (0.1 cm), and perform pulsed electroporation at 1.8 kV for 5 ms. Immediately after electroporation, add 1 mL of LBHIS medium to the cuvette, mix well, and transfer to a 1.5 mL centrifuge tube.

[1193] 6. Incubate at 30 °C for 1 hour.

[1194] 7. Finally, spread on a brain heart infusion agar plate containing 50 ug / mL kanamycin.

[1195] 8. Add the frozen or stab-cultured Corynebacterium glutamicum constructed in "7" or the Corynebacterium glutamicum containing the building blocks in "7" into a brain heart infusion liquid medium without antibiotics, where the volume of the medium does not exceed 1 / 5 of the conical flask volume, and culture at 30°C and 220 rpm.

[1196] 9. Add an IPTG inducer with a final concentration of 1 mM to the medium described in "8".

[1197] 10. After overnight induction (18 h) of Corynebacterium glutamicum, streak it on a solid LB medium plate without antibiotics.

[1198] 11. Pick the single colonies obtained in "10" and spot them on solid medium plates with and without antibiotics respectively (each single colony needs to be spotted twice to ensure the distinction of phenotypic differences).

[1199] 12. The specific results are as Figure 3 shown. After plasmid elimination, most of the cultured bacteria lost their resistance to antibiotics and could no longer grow on the antibiotic-containing plates, indicating that the relevant resistance genes have been lost and the plasmid has been successfully eliminated. Inoculate the corresponding strain with successful elimination to obtain the strain with successfully eliminated plasmid.

[1200] Example 8: Plasmid elimination using a constitutive promoter of Saccharomyces cerevisiae + LacI repressor protein plasmid elimination module

[1201] First, prepare the following reagents:

[1202] 10*TE buffer

[1203] 100 mM LiAC buffer

[1204] 1 M DTT buffer

[1205] ddH2O

[1206] 1 M sorbitol solution

[1207] Preparation of Saccharomyces cerevisiae competent cells:

[1208] 1. Pre-cool the reagents.

[1209] 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.

[1210] 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.

[1211] 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 100 mM LiAC buffer, and 0.5 mL of 1 M DTT buffer. Pipette to mix well, and place the centrifuge tube in a constant temperature water bath shaker at 30 °C and 50 r×min-1 for 45 min.

[1212] 5. Add 13.5 mL of pre-cooled ddH2O to a 50 mL centrifuge tube, centrifuge at 5000 r×min-1 in a centrifuge at 4 °C for 5 min, discard the supernatant, and collect the cells.

[1213] 6. Add 25 mL of pre-cooled ddH2O to a 50 mL centrifuge tube, pipette to resuspend the cells, centrifuge at 5000 r×min-1 in a centrifuge for 5 min, discard the supernatant, and collect the cells. Subsequently, wash the cells twice with 1 M sorbitol (25 mL for the first time and 20 mL for the second time).

[1214] 7. Add 1 mL of pre-cooled sorbitol to the centrifuge tube to resuspend the cells, and dispense them into 1.5 mL EP tubes, 80 μL per tube, for immediate use.

[1215] 8. During electroporation, place the dispensed competent cells on ice and add 5 μL of the plasmid obtained in Example 4.

[1216] 9. Transfer the mixture to a cold electroporation cuvette (0.2 cm), and perform pulsed electroporation at 1.5 kV, 200 Ω, 25 uF, and 5 ms. Immediately after electroporation, add 1 mL of antibiotic-free YPD medium to a 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.

[1217] 10. Centrifuge to collect the cells. Take 150 μL of the cell suspension and spread it on a resistant plate, and culture at 30 °C for 2 - 3 days.

[1218] 11. Add the cryopreserved cells or stab cultures of Saccharomyces cerevisiae constructed in "10" or Saccharomyces cerevisiae containing the building blocks constructed in "10" to an antibiotic-free YPD liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, and incubate at 30 °C and 220 rpm.

[1219] 12. When the OD600 of the cell suspension in "11" reaches 2, add an IPTG inducer with a final concentration of 1 mM.

[1220] 13. After inducing Saccharomyces cerevisiae for 48 h, streak it on an antibiotic-free LB solid medium plate.

[1221] 14. Take the single colonies obtained in "13" and spot them on antibiotic-free and antibiotic-containing solid medium plates respectively (each single colony needs to be spotted on the plate twice to ensure the distinction of phenotypic differences).

[1222] 15. The specific results are as Figure 4 shown. After plasmid elimination, most of the growing bacteria lost their resistance to antibiotics 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 corresponding strain with successful elimination can obtain the strain with successfully eliminated plasmid.

[1223] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by using equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A repressor-based plasmid elimination module, characterized in that, It includes two types of independent expression regions. One type of region is the repressor gene transcription region, which consists of the constitutive promoter carried by the repressor gene, including its RNA polymerase binding site, transcription start site, ribosome binding site, repressor translation region, and terminator; the other type of region is the antisense RNA expression region, which consists of a constitutive promoter, a repressor binding site, antisense RNA, and a terminator; the antisense RNA is the antisense RNA of the metabolic key gene mRNA and can bind to the metabolic key gene mRNA to hinder the translation of the metabolic key gene mRNA.

2. The plasmid elimination module according to claim 1, characterized in that, The repressor protein can bind to a specific DNA sequence, inhibit the transcription of downstream DNA after binding, be activated or released from the binding activity by a specific compound, and does not affect the growth of the chassis cell. The repressor binding site binds to the repressor protein and hinders the RNA polymerase reaction from passing through or transcribing the DNA sequence downstream of the binding site across the binding site.

3. The plasmid elimination module according to claim 2, wherein The repressor gene is the LacI protein gene, and the repressor binding site is the LacI protein binding site.

4. The plasmid elimination module according to claim 1, wherein: The constitutive promoter is a specific DNA sequence that can bind to RNA polymerase in the cell with a certain strength and initiate the transcription function of RNA polymerase to transcribe the downstream functional sequence and antisense RNA.

5. The plasmid elimination module according to claim 1, wherein: The constitutive promoter includes T7, tet, trc, trp, J23119 promoter, tac, and the constitutive promoters related to the common antibiotics chloramphenicol, kanamycin, and penicillin resistance genes of Corynebacterium glutamicum.

6. The plasmid elimination module according to claim 1, characterized in that, The antisense RNA expression region is carried on the plasmid to be eliminated.

7. The plasmid elimination module according to claim 1, wherein The plasmid to be eliminated is a circular or linear DNA that can replicate independently outside the genome stably in the cell or replicate with the genome and be passed on to daughter cells.

8. The plasmid elimination module according to claim 7, wherein The plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and yeast 2μ.

9. The plasmid elimination module according to claim 1, characterized in that, The gene interfered by the antisense RNA is the antisense RNA of the metabolic key gene mRNA and can bind to the metabolic key gene mRNA to hinder the translation of the metabolic key gene mRNA.

10. The plasmid elimination module according to claim 9, wherein The key metabolic genes are genes related to the TCA cycle, genes related to protein synthesis, genes related to aerobic respiration, genes related to anaerobic respiration, genes related to lipid synthesis, genes related to cell wall synthesis, genes related to sugar transport pathways, genes related to amino acid transport pathways, genes related to ATP synthesis, genes related to cytoskeleton synthesis, genes related to NADPH / NADP + synthesis, genes related to NADH / NAD + synthesis, genes related to vitamin synthesis, genes related to one-carbon transport systems, genes related to carbon dioxide fixation, genes related to DNA / RNA synthesis, genes related to amino acid synthesis, genes related to ribose synthesis, genes related to ribosome synthesis, genes related to ribosome assembly, genes related to aminoacyl-tRNA synthetase, and isozyme genes of the above genes.

11. The plasmid elimination module according to any one of claims 1 to 10, characterized in that, The elimination target of the plasmid elimination module is all microorganisms, animals, or plant cells that have stably free plasmids, have a probability of producing plasmid-free daughter cells during the proliferation process, and the elimination of free plasmids will not cause cell death or inability to proliferate.

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