Tandem expression module based on ribosome recovery factor antisense RNA

Abstract

The invention relates to the field of gene engineering, in particular to a tandem expression module based on ribosome recovery factor antisense RNA, which comprises two mutually independent expression regions, one region is an inducible factor transcription region and comprises a constitutive promoter of an inducible transcription factor fragment, and the other region is a non-constitutive promoter of an inducible transcription factor fragment. The kit comprises an RNA polymerase binding site, a transcription start site, a ribosome binding site, an induced transcription factor translation region and a terminator, the other region is an frr gene antisense RNA transcription region and comprises an arabinose transcription factor binding site, an frr antisense RNA transcription region and a terminator; in addition, two specific linker sequences linking different protein translation regions in tandem expression are included. The combination of the antisense RNA sequence, the expression structure of the antisense RNA sequence and the linker sequence between the expression cassettes can effectively adjust the relative expression proportion of tandem expression upstream and downstream proteins, and is used for gene function circuits with related requirements and optimal expression proportion exploration for specific protein pairs.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology, and is a combination of synthetic biology sequence elements with regulatory functions. It can regulate the expression ratio of the expression cassette downstream of the linker sequence relative to the upstream expression cassette to meet the control requirements of the relative expression levels of different proteins under different background conditions and expression purposes. Background Art

[0002] The tandem expression module of expression cassettes has been involved in many documents, but there is no literature that has systematically studied or sorted out the linker sequences used in the tandem expression module of expression cassettes and their cell background regulation strategies.

[0003] Existing tandem expression modules based on expression cassettes basically do not involve the regulation of the relative expression levels of tandem-expressed proteins. Most tandem expression technologies can only express different proteins in the tandem expression cassette at a specific relative expression level, and can only be used for specific experimental projects and specific experimental purposes, lacking adjustability and universality.

[0004] If a tandem expression module with adjustable relative expression levels can be found, it can significantly reduce the cost of exploring the optimal relative expression ratio. At the same time, the tandem expression module with adjustable relative expression levels itself can be used as a regulatory element in synthetic biology-related research and production. Summary of the Invention

[0005] The present invention proposes a set of tandem expression modules for expression cassettes, including the linker sequences used and the regulatory gene circuit for the relative expression levels of tandem-expressed proteins. The combination of the linker sequence and the regulatory gene circuit for the relative expression levels of tandem-expressed proteins can effectively regulate the relative protein expression levels of adjacent expression cassettes, improving the universality of the tandem expression technology for expression cassettes and laying a good foundation for further constructing a general tandem expression technology.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] According to an embodiment of the present invention, a tandem expression module based on the antisense RNA of ribosome recycling factor is characterized in that it includes two independent expression regions. One region is the transcription factor transcription region, including the constitutive promoter carried by the transcription factor fragment, which includes an RNA polymerase binding site, a transcription start site, a ribosome binding site, a transcription factor translation region, and a terminator; the other region is the frr gene antisense RNA transcription region, including a transcription factor binding site, an frr antisense RNA transcription region, and a terminator; it also includes a specific linker sequence that connects different protein translation regions in tandem expression.

[0008] According to an embodiment of the present invention, the transcription factor is a protein that can induce the transcription of genes downstream of the binding site after binding to a specific compound; or a protein that can induce the transcription of downstream genes before binding to a specific compound and cannot induce the transcription of genes downstream of the binding site after binding to the specific compound.

[0009] According to an embodiment of the present invention, the transcription factor is an arabinose transcription factor.

[0010] According to an embodiment of the present invention, the constitutive promoter carried by the transcription factor fragment is the constitutive promoter carried by the arabinose transcription factor.

[0011] According to an embodiment of the present invention, the tandem expression module is carried on a vector plasmid, a genome, or a free circular / linear mRNA.

[0012] According to an embodiment of the present invention, the vector plasmid is an Escherichia coli plasmid, including pet28a.

[0013] According to an embodiment of the present invention, the linker sequence is TGATG.

[0014] According to an embodiment of the present invention, the tandem expression module is used to simultaneously and efficiently translate multiple target proteins and regulate the relative expression ratios of proteins upstream and downstream of the linker sequence.

[0015] For the convenience of those skilled in the art to understand the present invention, the main vocabulary explanations are as follows:

[0016] Gene circuit: A combination of DNA sequences that combines specific gene elements such as promoters, terminators, protein translation regions, and specific protein binding regions in a single-site or multi-site arrangement according to certain rules to achieve specific functions.

[0017] Universality: This module can be used across species in the form of codon optimization / replacement of related control elements.

[0018] Escherichia coli ribosome recycling factor frr expression frame sequence:

[0019] GTGATTAGCGATATCAGAAAAGATGCTGAAGTACGCATGGACAAATGCGTAGAAGCGTT

[0020] CAAAACCCAAATCAGCAAAATACGCACGGGTCGTGCTTCTCCCAGCCTGCTGGATGGCA

[0021] TTGTCGTGGAATATTACGGCACGCCGACGCCGCTGCGTCAGCTGGCAAGCGTAACGGTA

[0022] GAAGATTCCCGTACACTGAAAATCAACGTGTTTGATCGTTCAATGTCTCCGGCCGTTGAA

[0023] AAAGCGATTATGGCGTCCGATCTTGGCCTGAACCCGAACTCTGCGGGTAGCGACATCCG

[0024] TGTTCCGCTGCCGCCGCTGACGGAAGAACGTCGTAAAGATCTGACCAAAATCGTTCGTG

[0025] GTGAAGCAGAACAAGCGCGTGTTGCAGTACGTAACGTGCGTCGTGACGCGAACGACAA

[0026] AGTGAAAGCACTGTTGAAAGATAAAGAGATCAGCGAAGACGACGATCGCCGTTCTCAG

[0027] GACGATGTACAGAAACTGACTGATGCTGCAATCAAGAAAATTGAAGCGGCGCTGGCAGACAAAGAAGCAGAACTGATGCAGTTCTGA(SEQ ID NO:1)

[0028] Full sequence of pET28a plasmid:

[0029] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGC

[0030] GCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT

[0031] CCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAG

[0032] GGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTT

[0033] CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGT

[0034] TCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT

[0035] CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA

[0036] ACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTT

[0037] TCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT

[0038] CCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATT

[0039] CATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA

[0040] CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCG

[0041] TCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAA

[0042] ATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCA

[0043] GACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAAC

[0044] CGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC

[0045] AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATA

[0046] TTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAG

[0047] TGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGC

[0048] ATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTAC

[0049] CTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTG

[0050] TCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCAT

[0051] GTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACC

[0052] CCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACG

[0053] TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG

[0054] ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCG

[0055] GTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

[0056] CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA

[0057] GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC

[0058] CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG

[0059] CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA

[0060] CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

[0061] GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACG

[0062] AGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT

[0063] CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG

[0064] CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT

[0065] TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATAC

[0066] CGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA

[0067] GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGT

[0068] GCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATC

[0069] GCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCT

[0070] GACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGC

[0071] TGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAG

[0072] CTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTC

[0073] GTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGC

[0074] GGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGT

[0075] AATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATG

[0076] CCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCA

[0077] GAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCAC

[0078] AGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGAC

[0079] TTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCA

[0080] GGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT

[0081] CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACG

[0082] ATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACG

[0083] TTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACC

[0084] GCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGA

[0085] CCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGT

[0086] GCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTC

[0087] TCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGT

[0088] TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0089] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA

[0090] CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG

[0091] CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACG

[0092] GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACC

[0093] AACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG

[0094] CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0095] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTG

[0096] AGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC

[0097] TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTAC

[0098] CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAAT

[0099] AACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0100] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC

[0101] AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0102] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG

[0103] TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA

[0104] ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG

[0105] CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGAC

[0106] ATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT

[0107] CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0108] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG

[0109] CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGG

[0110] GCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGA

[0111] TCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGT

[0112] GATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAAT

[0113] ACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT

[0114] TAACTTTAAGAAGGAGATATACCATGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC

[0115] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:2)

[0116] J23119 promoter:

[0117] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC→(SEQ ID NO:3)

[0118] rrnB T1 terminator:

[0119] CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC(SEQ ID NO:4)

[0120] Arabinose promoter sequence (arrow indicates the transcription start direction):

[0121] AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT

[0122] TCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACC

[0123] AAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACA

[0124] TTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ ID NO: 5) T7 terminator sequence (the arrow indicates the transcription termination direction):

[0125] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO: 6) Arabinose transcription factor expression cassette (including promoter, CDS region, terminator):

[0126] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACC

[0127] TGCATCGATTTATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCA

[0128] CGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTG

[0129] ATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAA

[0130] GCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACT

[0131] GCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCT

[0132] GGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTAC

[0133] CCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAA

[0134] TTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTT

[0135] AATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGA

[0136] ACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGA

[0137] CGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATG

[0138] AATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCT

[0139] GATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCA

[0140] GCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCC

[0141] ACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATA

[0142] CTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGC

[0143] CGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAA

[0144] AGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCT(SEQ ID NO:7)

[0145] T7 terminator sequence (the arrow indicates the transcription termination direction):

[0146] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO: 8) Human CALCA related gene sequence:

[0147] ATGGGCTTTCAGAAATTTAGCCCGTTTCTGGCGCTGAGCATTCTGGTGCTGCTGCAGGCG

[0148] GGCAGCCTGCATGCGGCGCCGTTTCGCAGCGCGCTGGAAAGCAGCCCGGCGGATCCGG

[0149] CGACCCTGAGCGAAGATGAAGCGCGCCTGCTGCTGGCGGCGCTGGTGCAGGATTATGTG

[0150] CAGATGAAAGCGAGCGAACTGGAACAGGAACAGGAACGCGAAGGCAGCCGCATTATTG

[0151] CGCAGAAACGCGCGTGCGATACCGCGACCTGCGTGACCCATCGCCTGGCGGGCCTGCT

[0152] GAGCCGCAGCGGCGGCGTGGTGAAAAATAATTTTGTGCCGACCAATGTGGGCAGCAAAGCGTTTGGCCGCCGCCGCCGCGATCTGCAGGCGTGA(SEQ ID NO: 9)

[0153] Human ITGB1BP2 related gene sequence:

[0154] ATGGCGCTGGAACAGAAAGAACTGGATCAGGAACCGGGCGCGGGCCTGGATAGCCTGA

[0155] TTCGCACCGGCAGCAGCTGCCAGAATCCGGGCTGCGATGCGGTGTATCAGGGCCCGGA

[0156] AAGCGATGCGACCCCGTGCACCTATCATCCGGGCGCGCCGCGCTTTCATGAAGGCATGA

[0157] AAAGCTGGAGCTGCTGCGGCATTCAGACCCTGGATTTTGGCGCGTTTCTGGCGCAGCCG

[0158] GGCTGCCGCGTGGGCCGCCATGATTGGGGCAAACAGCTGCCGGCGAGCTGCCGCCATG

[0159] ATTGGCATCAGACCGATAGCCTGGTGGTGGTGACCGTGTATGGCCAGATTCCGCTGCCG

[0160] GCGTTTAATTGGGTGAAAGCGAGCCAGACCGAACTGCATGTGCATATTGTGTTTGATGG

[0161] CAATCGCGTGTTTCAGGCGCAGATGAAACTGTGGGGCGTGATTAATGTGGAACAGAGCA

[0162] GCGTGTTTCTGATGCCGAGCCGCGTGGAAATTAGCCTGGTGAAAGCGGATCCGGGCAGC

[0163] TGGGCGCAGCTGGAACATCCGGATGCGCTGGCGAAAAAAGCGCGCGCGGGCGTGGTGC

[0164] TGGAAATGGATGAAGAAGAAAGCGATGATAGCGATGATGATCTGAGCTGGACCGAAGAAGAAGAAGAAGAAGCGATGGGCGAATAG(SEQ ID NO:10)

[0165] When ribosomes encounter a stop codon during transcription on an mRNA with tandem expression frames, there may be two situations:

[0166] 1. Ribosomes are depolymerized into free ribosomal subunits by ribosome recycling factors and bind to the RBS sequence on another mRNA to initiate a new round of translation.

[0167] 2. Ribosomes continue to slide towards the 3'-end of the mRNA and resume translation after encountering the start codon of a new expression frame.

[0168] The beneficial effects of the present invention are as follows: The present invention proposes an interfering sequence against the frr gene of Escherichia coli ribosome recycling factor and a combined application method based on the linker sequence between frr and a specific expression frame.

[0169] The combination of the antisense RNA sequence designed based on the present invention, its expression structure, and the linker sequence between expression frames can effectively regulate the relative expression ratio of upstream and downstream proteins expressed in tandem, and is used for gene function circuits with relevant requirements and the exploration of the optimal expression ratio for specific protein pairs; the design proposed by the present invention can be used as a regulatory element in synthetic biology-related research and production. It also provides the possibility for constructing more complex gene function circuits in the future. Specific Embodiments

[0170] The present invention will be specifically introduced below in conjunction with specific embodiments.

[0171] Instrumentation and Equipment:

[0172] 1. PCR instrument

[0173] 2. Electrophoresis instrument

[0174] 3. Electrophoresis tank

[0175] 4. Ultra-low temperature refrigerator

[0176] 5. Biochemical incubator

[0177] 6. Autoclave

[0178] 7. Water bath

[0179] 8. Medical freezer

[0180] 9. Tabletop centrifuge

[0181] 10. Pipettor

[0182] 11. Oven

[0183] 12. Youmi microplate reader

[0184] Reagents:

[0185]

[0186]

[0187] Biological Materials:

[0188] Related Primers and Synthetic Sequences:

[0189] EP1: CATGTTGTTGCTCAGGTCGCAGACG (SEQ ID NO: 11)

[0190] EP2: GGAAGGGAAGAAAGCGAAAGGAGC (SEQ ID NO: 12)

[0191] EP3: CCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCG (SEQ ID NO: 13)

[0192] EP4: CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTC (SEQ ID NO: 14)

[0193] EP5: GATCCGGAACATAATGGTGCAGG (SEQ ID NO: 15)

[0194] EP6: GCACTAAATCGGAACCCTAAAGG (SEQ ID NO: 16)

[0195] Sequences synthesized by commissioning (first ITGB1BP2, then CALCA):

[0196] CATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTT

[0197] CGCTCGCGTATCGGTGATTCATTCGAAGAAAGGCCCACCCGTGAAGGTGA

[0198] GCCTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCA

[0199] CGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAA

[0200] TAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCAT

[0201] CCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGC

[0202] GCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGA

[0203] CGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATT

[0204] GCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATT

[0205] ATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAA

[0206] CAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCC

[0207] TTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTG

[0208] CGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTT

[0209] AAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGAT

[0210] ACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCG

[0211] GGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTT

[0212] TCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTC

[0213] CCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGC

[0214] GTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGG

[0215] ATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAA

[0216] ACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGC

[0217] TCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAAC

[0218] AAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAAT

[0219] CACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTAT

[0220] GCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATC

[0221] GCAACTCTCTACTGTTTCTCCATCAGTGTACGGGAATCTTCTACCGTTACGC

[0222] TTGCCAGCTGACGCAGCGCTAGCATAACCCCTTGGGGCCTCTAAACGGGT

[0223] CTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATCT

[0224] AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTCATG

[0225] CCTGCAGATCGCGGCGGCGGCGGCCAAACGCTTTGCTGCCCACATTGGTC

[0226] GGCACAAAATTATTTTTCACCACGCCGCCGCTGCGGCTCAGCAGGCCCGC

[0227] CAGGCGATGGGTCACGCAGGTCGCGGTATCGCACGCGCGTTTCTGCGCAAT

[0228] AATGCGGCTGCCTTCGCGTTCCTGTTCCTGTTCCAGTTCGCTCGCTTTCATC

[0229] TGCACATAATCCTGCACCAGCGCCGCCAGCAGCAGGCGCGCTTCATCTTCG

[0230] CTCAGGGTCGCCGGATCCGCCGGGCTGCTTTCCAGCGCGCTGCGAAACGG

[0231] CGCCGCATGCAGGCTGCCCGCCTGCAGCAGCACCAGAATGCTCAGCGCCA

[0232] GAAACGGGCTAAATTTCTGAAAGCCCATCATCATCATTCGCCCATCGCTTCT

[0233] TCTTCTTCTTCTTCTTCGGTCCAGCTCAGATCATCATCGCTATCATCGCTTTC

[0234] TTCTTCATCCATTTCCAGCACCACGCCCGCGCGCGCTTTTTTCGCCAGCGC

[0235] ATCCGGATGTTCCAGCTGCGCCCAGCTGCCCGGATCCGCTTTCACCAGGCT

[0236] AATTTCCACGCGGCTCGGCATCAGAAACACGCTGCTCTGTTCCACATTAAT

[0237] CACGCCCCACAGTTTCATCTGCGCCTGAAACACGCGATTGCCATCAAACAC

[0238] AATATGCACATGCAGTTCGGTCTGGCTCGCTTTCACCCAATTAAACGCCGG

[0239] CAGCGGAATCTGGCCATACACGGTCACCACCACCAGGCTATCGGTCTGATG

[0240] CCAATCATGGCGGCAGCTCGCCGGCAGCTGTTTGCCCCAATCATGGCGGCC

[0241] CACGCGGCAGCCCGGCTGCGCCAGAAACGCGCCAAAATCCAGGGTCTGA

[0242] ATGCCGCAGCAGCTCCAGCTTTTCATGCCTTCATGAAAGCGCGGCGCGCCC

[0243] GGATGATAGGTGCACGGGGTCGCATCGCTTTCCGGGCCCTGATACACCGCA

[0244] TCGCAGCCCGGATTCTGGCAGCTGCTGCCGGTGCGAATCAGGCTATCCAG

[0245] GCCCGCGCCCGGTTCCTGATCCAGTTCTTTCTGTTCCAGCGCCATGCTAGC

[0246] ATTATACCTAGGACTGAGCTAGCTGTCAAGCTCCTTTCGCTTTCTTCCCTTCC(SEQ ID NO:17)

[0247] 委托合成的序列2(先CALCA,后ITGB1BP2):

[0248] CATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTT

[0249] CGCTCGCGTATCGGTGATTCATTCGAAGAAAGGCCCACCCGTGAAGGTGA

[0250] GCCTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCA

[0251] CGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAA

[0252] TAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCAT

[0253] CCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGC

[0254] GCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGA

[0255] CGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATT

[0256] GCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATT

[0257] ATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAA

[0258] CAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCC

[0259] TTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTG

[0260] CGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTT

[0261] AAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGAT

[0262] ACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCG

[0263] GGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTT

[0264] TCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTC

[0265] CCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGC

[0266] GTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGG

[0267] ATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAA

[0268] ACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGC

[0269] TCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAAC

[0270] AAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAAT

[0271] CACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTAT

[0272] GCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATC

[0273] GCAACTCTCTACTGTTTCTCCATCAGTGTACGGGAATCTTCTACCGTTACGC

[0274] TTGCCAGCTGACGCAGCGCTAGCATAACCCCTTGGGGCCTCTAAACGGGT

[0275] CTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATCT

[0276] AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTATT

[0277] CGCCCATCGCTTCTTCTTCTTCTTCTTCTTCGGTCCAGCTCAGATCATCATC

[0278] GCTATCATCGCTTTCTTCTTCATCCATTTCCAGCACCACGCCCGCGCGCGCT

[0279] TTTTTCGCCAGCGCATCCGGATGTTCCAGCTGCGCCCAGCTGCCCGGATCC

[0280] GCTTTCACCAGGCTAATTTCCACGCGGCTCGGCATCAGAAACACGCTGCTC

[0281] TGTTCCACATTAATCACGCCCCACAGTTTCATCTGCGCCTGAAACACGCGA

[0282] TTGCCATCAAACACAATATGCACATGCAGTTCGGTCTGGCTCGCTTTCACC

[0283] CAATTAAACGCCGGCAGCGGAATCTGGCCATACACGGTCACCACCACCAG

[0284] GCTATCGGTCTGATGCCAATCATGGCGGCAGCTCGCCGGCAGCTGTTTGCC

[0285] CCAATCATGGCGGCCCACGCGGCAGCCCGGCTGCGCCAGAAACGCGCCAA

[0286] AATCCAGGGTCTGAATGCCGCAGCAGCTCCAGCTTTTCATGCCTTCATGAA

[0287] AGCGCGGCGCGCCCGGATGATAGGTGCACGGGGTCGCATCGCTTTCCGGG

[0288] CCCTGATACACCGCATCGCAGCCCGGATTCTGGCAGCTGCTGCCGGTGCGA

[0289] ATCAGGCTATCCAGGCCCGCGCCCGGTTCCTGATCCAGTTCTTTCTGTTCC

[0290] AGCGCCATCATCATCACGCCTGCAGATCGCGGCGGCGGCGGCCAAACGCT

[0291] TTGCTGCCCACATTGGTCGGCACAAAATTATTTTTCACCACGCCGCCGCTG

[0292] CGGCTCAGCAGGCCCGCCAGGCGATGGGTCACGCAGGTCGCGGTATCGCA

[0293] CGCGCGTTTCTGCGCAATAATGCGGCTGCCTTCGCGTTCCTGTTCCTGTTCC

[0294] AGTTCGCTCGCTTTCATCTGCACATAATCCTGCACCAGCGCCGCCAGCAGC

[0295] AGGCGCGCTTCATCTTCGCTCAGGGTCGCCGGATCCGCCGGGCTGCTTTCC

[0296] AGCGCGCTGCGAAACGGCGCCGCATGCAGGCTGCCCGCCTGCAGCAGCA

[0297] CCAGAATGCTCAGCGCCAGAAACGGGCTAAATTTCTGAAAGCCCATGCTA

[0298] GCATTATACCTAGGACTGAGCTAGCTGTCAAGGGACGCGCCCTGTAGCGGC

[0299] GCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCC(SEQ ID NO: 18)

[0300] Construction of RNA interference tandem expression plasmid of Escherichia coli frr protein in Example 1

[0301] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0302] 1. Design primers EP001 and EP002 to amplify two synthetic sequences (SEQ ID NO: 17 and SEQ ID NO: 18) according to the following system and PCR program. Design primers EP003 and EP004 to amplify the plasmid template pet28a plasmid according to the same system and PCR program.

[0303] System:

[0304]

[0305] PCR program:

[0306]

[0307] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products of the two synthetic sequences and the PCR product of the pet28a plasmid according to the purification kit instructions.

[0308] 3. Connect the PCR purified products of the two synthetic sequences and the PCR purified product of the pet28a plasmid respectively according to the operation procedure shown in the ready-to-use seamless cloning kit instructions.

[0309] 4. Transform according to the following steps Overnight culture after 5α Chemically Competent Cell:

[0310] 1) Take 100 μL of competent cells melted on ice, add the two ligation products in "3" respectively, mix gently (pipette gently or flick the tube wall several times), and let stand on ice for 5 min.

[0311] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0312] 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.

[0313] 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.

[0314] (2) Sequencing verification experiment for the construction result:

[0315] Take the single colonies obtained from the spread plate and send them for sequencing with primers EP005 and EP006 to verify the construction result.

[0316] Plasmid construction result:

[0317] Construction result of ITGB1BP2-CALCA expression plasmid:

[0318] TGGCGAATCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTCATGCCTGCAGATCGCGGCGGCGGCGGCCAAACGCTTTGCTGCCCACATTGGTCGGCACAAAATTATTTTTCACCACGCCGCCGCTGCGGCTCAGCAGGCCCGCCAGGCGATGGGTCA

[0319] CGCAGGTCGCGGTATCGCACGCGCGTTTCTGCGCAATAATGCGGCTGCCTTCGCGTTCCT

[0320] GTTCCTGTTCCAGTTCGCTCGCTTTCATCTGCACATAATCCTGCACCAGCGCCGCCAGCA

[0321] GCAGGCGCGCTTCATCTTCGCTCAGGGTCGCCGGATCCGCCGGGCTGCTTTCCAGCGCG

[0322] CTGCGAAACGGCGCCGCATGCAGGCTGCCCGCCTGCAGCAGCACCAGAATGCTCAGCG

[0323] CCAGAAACGGGCTAAATTTCTGAAAGCCCATCATCATCATTCGCCCATCGCTTCTTCTTC

[0324] TTCTTCTTCTTCGGTCCAGCTCAGATCATCATCGCTATCATCGCTTTCTTCTTCATCCATTT

[0325] CCAGCACCACGCCCGCGCGCGCTTTTTTCGCCAGCGCATCCGGATGTTCCAGCTGCGCC

[0326] CAGCTGCCCGGATCCGCTTTCACCAGGCTAATTTCCACGCGGCTCGGCATCAGAAACAC

[0327] GCTGCTCTGTTCCACATTAATCACGCCCCACAGTTTCATCTGCGCCTGAAACACGCGATT

[0328] GCCATCAAACACAATATGCACATGCAGTTCGGTCTGGCTCGCTTTCACCCAATTAAACGC

[0329] CGGCAGCGGAATCTGGCCATACACGGTCACCACCACCAGGCTATCGGTCTGATGCCAAT

[0330] CATGGCGGCAGCTCGCCGGCAGCTGTTTGCCCCAATCATGGCGGCCCACGCGGCAGCCC

[0331] GGCTGCGCCAGAAACGCGCCAAAATCCAGGGTCTGAATGCCGCAGCAGCTCCAGCTTT

[0332] TCATGCCTTCATGAAAGCGCGGCGCGCCCGGATGATAGGTGCACGGGGTCGCATCGCTT

[0333] TCCGGGCCCTGATACACCGCATCGCAGCCCGGATTCTGGCAGCTGCTGCCGGTGCGAAT

[0334] CAGGCTATCCAGGCCCGCGCCCGGTTCCTGATCCAGTTCTTTCTGTTCCAGCGCCATGCT

[0335] AGCATTATACCTAGGACTGAGCTAGCTGTCAAGCTCCTTTCGCTTTCTTCCCTTCCTTTCT

[0336] CGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCC

[0337] GATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTA

[0338] GTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTA

[0339] ATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTG

[0340] ATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAA

[0341] ATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGG

[0342] AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA

[0343] TGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAG

[0344] GATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGA

[0345] GGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACAT

[0346] CAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCAT

[0347] GAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTT

[0348] CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCA

[0349] TTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAA

[0350] ACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACC

[0351] TGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAG

[0352] TAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTC

[0353] CGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCC

[0354] ATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACC

[0355] TGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAA

[0356] TTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTA

[0357] TTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTT

[0358] TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

[0359] TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT

[0360] GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG

[0361] CAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT

[0362] GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC

[0363] GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG

[0364] GTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC

[0365] GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA

[0366] AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC

[0367] TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG

[0368] AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

[0369] GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGT

[0370] TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG

[0371] CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGAT

[0372] GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCA

[0373] GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGA

[0374] CTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT

[0375] TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTG

[0376] TCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAG

[0377] CGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTT

[0378] TCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTT

[0379] CCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATAC

[0380] CGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTA

[0381] CTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAA

[0382] TCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGC

[0383] CAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGT

[0384] TTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAG

[0385] ACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCGAAGAA

[0386] AGGCCCACCCGTGAAGGTGAGCCTTATGACAACTTGACGGCTACATCATTCACTTTTTCT

[0387] TCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGA

[0388] GAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGG

[0389] TGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACG

[0390] CTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACAT

[0391] GCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGAC

[0392] AAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGC

[0393] GCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCC

[0394] CTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCAT

[0395] CCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAG

[0396] TAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACG

[0397] ACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAA

[0398] ATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAAC

[0399] CTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGC

[0400] GTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTG

[0401] CGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTG

[0402] CATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAAC

[0403] CCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTA

[0404] ACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACA

[0405] CTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATC

[0406] GCAACTCTCTACTGTTTCTCCATCAGTGTACGGGAATCTTCTACCGTTACGCTTGCCAGC

[0407] TGACGCAGCGCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:19)

[0408] Results of construction of CALCA-ITGB1BP2 expression plasmid:

[0409]

[0410] GGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC

[0411] AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATG

[0412] GCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCAT

[0413] CAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA

[0414] AATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAG

[0415] GAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTG

[0416] GAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGAT

[0417] AAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTC

[0418] ATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATC

[0419] GGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCA

[0420] TTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTT

[0421] TCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTA

[0422] TTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA

[0423] GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA

[0424] ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT

[0425] TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTA

[0426] GCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT

[0427] AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC

[0428] AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA

[0429] CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG

[0430] AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG

[0431] GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT

[0432] AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGG

[0433] GGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG

[0434] CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT

[0435] ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGT

[0436] CAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGC

[0437] GGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT

[0438] AAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCC

[0439] GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC

[0440] AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAA

[0441] CGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTC

[0442] TGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCT

[0443] GATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTA

[0444] AGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGA

[0445] TACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTG

[0446] GCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCG

[0447] TTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGG

[0448] AACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACC

[0449] GAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACG

[0450] TTCGCTCGCGTATCGGTGATTCATTCGAAGAAAGGCCCACCCGTGAAGGTGAGCCTTAT

[0451] GACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGG

[0452] GCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAA

[0453] CATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGG

[0454] CTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAG

[0455] ATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAA

[0456] TTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATC

[0457] GGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAG

[0458] ATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCC

[0459] AAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGG

[0460] CAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACC

[0461] CACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGC

[0462] GGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCAC

[0463] CCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGAT

[0464] AAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCAT

[0465] TAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCC

[0466] CGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCT

[0467] TTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAAC

[0468] AAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAG

[0469] AAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCC

[0470] ATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATCAG

[0471] TGTACGGGAATCTTCTACCGTTACGCTTGCCAGCTGACGCAGCGCTAGCATAACCCCTTG

[0472] GGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:20)

[0473] (3) Plasmid extraction experiment:

[0474] 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.

[0475] Example 2 Effect of Frr protein RNA interference on protein expression of Escherichia coli MG1655 pet28a plasmid

[0476] (1) Plasmid transformation experiment

[0477] 1) Prepare competent cells from Escherichia coli MG1655 strain according to the procedure described in the Super Competent Cell Preparation Kit.

[0478] 2) Take 100 μL of competent cells melted on ice, add the ITGB1BP2-CALCA expression plasmid and CALCA-ITGB1BP2 expression plasmid constructed by the technical solution, mix gently (pipette gently or flick the tube wall several times), and let it stand on ice for 5 min.

[0479] 3) Heat shock in a 42°C water bath for 45 - 60 s, then 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).

[0480] 4) Add 700 μL of antibiotic-free sterile liquid medium (SOB or LB) to the centrifuge tube and mix well.

[0481] 5) Take an appropriate volume of the recovery solution and evenly spread it on an LB solid medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37°C incubator overnight.

[0482] (2) Shake flask fermentation experiment

[0483] 1) Take the single colony obtained in (4) and activate it overnight at 37°C in 3 mL of LB solution containing 50 μg / mL kanamycin.

[0484] 2) Inoculate 1% into six 250 mL sterile conical flasks containing 30 mL of LB liquid medium containing 50 μg / mL kanamycin, and after culturing for 3 h, add sterilized arabinose with final concentrations of 0%, 1%, 2%, 3%, 4%, and 5% respectively to induce for 6 h.

[0485] (3) Fermentation result processing

[0486] Take the fermentation results and extract the results according to the BeyoLytic TM Bacterial active protein extraction reagent according to the instructions.

[0487] Dilute the extracted results by *10, *100, *1000, *10000, *1000000.

[0488] (4) Elisa result detection

[0489] Take 1 μL of the diluted result and perform elisa detection according to the instructions of the Human ITGB1BP2 (Integrin beta-1-binding protein2) ELISA Kit and the Human CALCA (Calcitonin gene-related peptide 1) ELISA Kit respectively.

[0490] The results are as follows:

[0491]

[0492]

[0493]

[0494] Example 3

[0495]

[0496]

[0497]

[0498]

[0499] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A tandem expression module based on ribosome recycling factor antisense RNA, characterized in that, It includes two independent expression regions. One region is the transcription factor transcription region, which includes the constitutive promoter carried by the transcription factor fragment, and it includes an RNA polymerase binding site, a transcription start site, a ribosome binding site, a transcription factor translation region, and a terminator. The other region is the frr gene antisense RNA transcription region, which includes a transcription factor binding site, an frr antisense RNA transcription region, and a terminator. It also includes a specific linker sequence that connects different protein translation regions in tandem expression.

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

3. The tandem expression module according to claim 2, wherein The transcription factor is an arabinose transcription factor.

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

5. The tandem expression module according to claim 1, wherein The tandem expression module is carried on a vector plasmid, a genome, or free circular / linear mRNA.

6. The tandem expression module according to claim 5, wherein The vector plasmid is an Escherichia coli plasmid, including pet28a.

7. The tandem expression module according to claim 1, wherein The linker sequence is TGATG.

8. The tandem expression module according to any one of claims 1 to 7, characterized in that The tandem expression module is used to simultaneously and efficiently translate multiple target proteins and regulate the relative expression ratio of proteins upstream and downstream of the linker sequence.

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