Gene time-series expression regulation system and method based on single-base editing and application thereof

Abstract

The application discloses a gene timing expression regulation system and method based on single base editing and application thereof. A tandem DNA sequence recognized by ABE7.10 is constructed as a gene expression regulation switch element. Single base editing is performed on several adenine target points on the mutually influencing and tandem arranged DNA sequence by ABE7.10. Finally, the promoter -35 box sequence is modified to realize the closing or opening of the promoter overlapping the last target point, so as to achieve the purpose of delaying the activation or suppression of the expression of the target gene. The application can control the time of the expression of one or more genes by changing the number (length) of the fuse DNA sequence. The more the adenine target points, the longer the time required for the regulation of the target gene, so as to realize the pre-set timing regulation of gene expression.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to gene temporal expression regulation systems and methods based on single-base editing and their applications. Background Technology

[0002] The dynamic regulation of gene expression is an important area of ​​research in synthetic biology. Gene expression regulation can be achieved by altering cis-regulatory elements through genome editing.

[0003] The single-base editing system is based on the Cas9 (dCas9) and Cas9 cleavage enzyme (nCas9) which lack nuclease activity. It comprises two fusion proteins with single-base editing capabilities, obtained by David Liu's laboratory through phage-directed evolution: the cytosine base editor (CBE) and the adenine base editor (ABE). In the single-base editing system, dCas9 or nCas9 lacks the nuclease activity to cut double-stranded DNA, but retains DNA-binding activity. Without breaking the DNA double strand and providing a DNA template, sgRNA guides dCas9 or nCas9 in the fusion deaminase protein to bind to the target site through complementary pairing, directly and precisely editing the target site. This achieves single-base conversion from cytosine (C) to thymine (T) or guanine (G) to adenine (A) within a certain activity window. Among them, CBE can edit cytosine (C) in DNA double-stranded target sites into thymine (T), converting C·G into T·A; ABE can edit adenine (A) in DNA double-stranded target sites into guanine (G), converting A·T into G·C.

[0004] ABE is a fusion protein of *Escherichia coli* adenine deaminase and *Streptococcus pyogenes* nCas9 protein, evolved via phage-assisted evolution (Gaudelli NM, Komor AC, Rees HA, et al., *Nature*, 2017, 551, 464-471). Taking ABE7.10 as an example, ABE7.10, a base editor derived from the directed evolution of a fusion protein of spdCas9 and TadA, is obtained by adding an inactive *E. coli* deaminase (TadA*) and an active *E. coli* deaminase (TadA) to the N-terminus of nCas9 (D10A) and optimizing the amino acid composition. The three proteins are linked by a flexible linker, and its function is to recognize proteins with an N-terminus at the 3' end via sgRNA. The target site of the GG sequence binds to the TadA binding single-stranded region (the original spacer homologous sequence), causing adenine (A) at positions 4-7 in the original spacer to deaminate into hypoxanthine (I). Hypoxanthine pairs with cytosine (C). Therefore, after subsequent DNA replication, the original adenine (A) position is replaced by guanine (G), which pairs with cytosine (C). Simultaneously, the other strand of the double helix is ​​cleaved by the HNH domain of nCas9 (D10A), thus completing the A-to-G conversion. The basic function of ABE7.10 is to modify adenine to guanine in the 4-7 bases starting from the 5' end of the original spacer; these 4-7 bases are called the edit window.

[0005] The recognition DNA sequence site of ABE consists of two parts: a 20bp protospacer and a protospacer adjacent motif (PAM) immediately adjacent to the 3' end of the protospacer. The overall target sequence is 5'-(N*20)(NGG)-3'. The protospacer can be directly used as the design template for the spacer of sgRNA. The spacer can differ from the protospacer by 2bp or less. Differences may reduce binding efficiency. If the PAM, i.e., the NGG sequence, is incorrect (GG is replaced with other bases), ABE will hardly be able to bind.

[0006] Because single-base editing systems can achieve efficient single-base substitutions without introducing double-strand breaks, avoiding the uncontrollable insertion or deletion mutations (indels) introduced during nonhomologous end-joining (NHEJ) in traditional CRISPR / Cas9, the emergence of single-base editing technology has promoted the effectiveness and scope of point mutation gene editing. However, there are currently no reports in the literature or patents regarding the use of ABE technology to achieve temporal regulation of gene expression. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a gene expression temporal regulation system, method, and application based on single-base editing. This invention constructs a gene expression regulatory switch element, which uses ABE7.10 to perform single-base editing on several adenine target sites on a DNA sequence that are mutually influential and arranged in tandem. Finally, by modifying the -35box sequence of the promoter, the promoter overlapping the last target site is turned off or on, thereby achieving the purpose of delayed regulation of the activation or inhibition of the target gene expression.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention discloses a gene expression regulatory switch element, the overall organization (or overall sequence) of which is as follows:

[0010] 5'-Tac promoter-connector segment-[basic unit]×n-NBB-TGG-3'

[0011] in,

[0012] The sequence of the basic unit is: 5'-NBBTAABNNNNNNNNNN-3';

[0013] The sequence of the linker is: 5'-NNNNNNNNNNN-3';

[0014] The nucleotide sequence of the Tac promoter is shown in SEQ ID NO.1, and the -35box of the Tac promoter is TGTCAA;

[0015] B represents one of the bases G, T, and C; N represents any base; and n is the number of basic units, n = 1, 2, 3, ...

[0016] Let x be the sum of the number of G and C in the three bases closest to the 5' end of the basic unit, and y be the sum of the number of G and C in all 17 bases of the basic unit, then x + y = 10; let z be the number of G and C in all 11 bases of the linker segment, then x + z = 8.

[0017] In some specific embodiments of the present invention, the gene expression regulatory switch element has one initial transduction target site, m transduction target sites, and one activation target site in the initial state, where m = 0, 1, 2, 3...; wherein,

[0018] The initial transduction target point is located in the portion of the overall sequence closest to the 3' end, and the sequence of the initial transduction target point is: 5'-NBB-TAABNNNNNNNNNNNBB-TGG-3';

[0019] The transduction target is located in the middle of the overall sequence, and the sequence of the transduction target is: 5'-NBB-TAABNNNNNNNNNNNBB-TAA-3';

[0020] The excitation target is located in the portion of the overall sequence near the Tac promoter, and the sequence of the excitation target is: 5'-TGTCAA-connector segment-NBB-TAA-3';

[0021] The initial transduction target and the transduction target share a single sgRNA, called the transduction sgRNA, and the sequence of the transduction sgRNA is: 5-NBBTAABNNNNNNNNNNNBB-gRNAscaffold-3';

[0022] The sgRNA used to stimulate the target site is called the stimulating sgRNA, and the sequence of the stimulating sgRNA is 5'-TGTCAA-connector-NBB-gRNAscaffold-3'.

[0023] In some specific embodiments of the present invention, the nucleotide sequence of the Tac promoter is shown in SEQ ID NO.1, the nucleotide sequence of the basic unit is shown in SEQ ID NO.2, and the nucleotide sequence of the linker segment is shown in SEQ ID NO.3; the overall organization (or overall sequence) of the gene expression regulatory switching element is as follows:

[0024] 5'-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTGACG-[AGCTAACTGCAGTCACT]×n-NBB-TGG-3'

[0025] Where B represents one of the bases G, T, and C, N represents any base, and n = 1, 2, 3, ...

[0026] In some specific embodiments of the present invention, the gene expression regulatory switch element has one initial transduction target site, m transduction target sites, and one activation target site in the initial state, where m = 0, 1, 2, 3...; wherein,

[0027] The initial transduction target is located in the portion of the overall sequence closest to the 3' end, and the nucleotide sequence of the initial transduction target is shown in SEQ ID NO.4; the transduction target is located in the middle of the overall sequence, and the nucleotide sequence of the transduction target is shown in SEQ ID NO.5, m = 0, 1, 2, 3...; the activation target is located in the portion of the overall sequence close to the Tac promoter, and the nucleotide sequence of the activation target is shown in SEQ ID NO.6;

[0028] The initial transduction target and the transduction target share a single sgRNA, referred to as the transduction sgRNA, the nucleotide sequence of which is shown in SEQ ID NO.7; the sgRNA sequence used for the activation target is the activation sgRNA, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0029] In the gene expression regulatory switch element of the present invention, only the initial transduction target can be recognized and edited by ABE7.10 in the initial state. However, after the initial transduction target is modified by ABE7.10, because the 5' end of the initial transduction target overlaps with the 3' end of the transduction target, the above modification activates the PAM of the transduction target. This transduction target is then transformed into the initial transduction target and modified again, activating the next transduction target, and so on. Each target in the entire sequence from the 3' end to the 5' end will be activated sequentially. Finally, the two outermost adenine bases of the -35box of the Tac promoter are modified, causing the Tac promoter to be inactivated, thereby achieving the purpose of regulating gene expression. The logic of sequential activation of each target is similar to a fuse; therefore, the gene expression regulatory switch element is named a "fuse sequence".

[0030] This invention also discloses a gene expression regulation system, comprising: pFuse plasmid and pACYC-ABE7.10(dCas9) plasmid, wherein,

[0031] The pFuse plasmid is obtained by cloning the gene expression regulatory switch element of a predetermined length into the pCDFDuet-1 plasmid to obtain the trigger plasmid; then cloning the target gene and the signaling sgRNA into the trigger plasmid to obtain the pFuse plasmid.

[0032] The pACYC-ABE7.10(dCas9) plasmid was obtained by the following method: the 840th residue of ABE7.10 was point-mutated from histidine to alanine to obtain ABE7.10(dCas9); then the ABE7.10(dCas9) and the activation sgRNA were cloned into pACYCDuet-1 to obtain the pACYC-ABE7.10(dCas9) plasmid.

[0033] In some specific embodiments of the present invention, the target gene includes a functional gene or a regulatory factor.

[0034] In some specific examples of the present invention, the target gene includes functional genes or regulatory factors of natural product anabolic pathways, antibiotic anabolic pathways, and environmental pollutant degradation pathways.

[0035] This invention also discloses a method for regulating gene expression, comprising:

[0036] (1) Design and synthesize the above-mentioned gene expression regulatory switch element of predetermined length, clone the gene expression regulatory switch element into pCDFDuet-1 plasmid to construct the fuse plasmid, clone the target gene and the signaling sgRNA into the fuse plasmid to obtain pFuse plasmid;

[0037] (2) The 840th residue of ABE7.10 was point-mutated from histidine to alanine to obtain ABE7.10(dCas9); then the ABE7.10(dCas9) and the activation sgRNA were cloned into pACYCDuet-1 to obtain the pACYC-ABE7.10(dCas9) plasmid.

[0038] (3) The pFuse plasmid and pACYC-ABE7.10(dCas9) plasmid were simultaneously transformed into recipient cells to regulate the expression of the target gene.

[0039] In some specific examples of the present invention, in step (3), the pFuse plasmid and the pACYC-ABE7.10(dCas9) plasmid are simultaneously transformed into Escherichia coli competent cells to regulate the expression of the target gene.

[0040] In some specific embodiments of the present invention, the target gene includes a functional gene or a regulatory factor.

[0041] In some specific examples of the present invention, the target gene includes functional genes or regulatory factors of natural product anabolic pathways, antibiotic anabolic pathways, and environmental pollutant degradation pathways.

[0042] This invention also discloses the applications of the above-mentioned gene expression regulation switching element or gene expression regulation system, including but not limited to: pre-programming for the temporal sequential expression of genes synthesizing different metabolite pathways in engineered strains; gene expression control for engineered strains synthesizing natural products and antibiotics; and gene expression control for engineered bacteria synthesizing environmental pollutant treatment.

[0043] In this invention, ABE7.10(dCas9) is also referred to as ABE7.10-dCas9, and the pACYC-ABE7.10(dCas9) plasmid is also referred to as pACYC-ABE7.10-dCas9 plasmid.

[0044] Compared with the prior art, the present invention has the following beneficial technical effects:

[0045] This invention constructs a tandem DNA sequence (named Fusesequence) recognized by the adenine base editing enzyme ABE7.10 as a gene expression regulatory switch element. ABE7.10 performs single-base editing on several adenine target sites on mutually influential, tandemly arranged DNA sequences. Finally, by modifying the -35box sequence of the promoter, the promoter overlapping the last target site is turned off or on, thereby achieving the purpose of delaying the activation or inhibition of the expression of the target gene.

[0046] This invention controls the timing of one or more gene expression events by changing the number of basic units (or the sequence length of the element) in the gene expression regulatory switching element. The more adenine target sites there are, the longer the generation time required for the target gene regulation to occur, thereby achieving pre-set gene expression timing regulation.

[0047] This invention is the first to realize a regulatory element that can pre-set gene expression timing, which can pre-set gene expression time in engineered bacteria. Its characteristics enable it to pre-program the temporal expression of genes synthesized by different metabolite pathways in engineered strains. It can be used for gene expression control in engineered strains for the synthesis of natural products and antibiotics in the biomedical field, as well as engineered strains for the treatment of environmental pollutants. Attached Figure Description

[0048] Figure 1 A schematic diagram illustrating the principle of activating adjacent conduction target points to transform the initial conduction target point in the fuse sequence into the initial conduction target point.

[0049] Figure 2 This is a schematic diagram illustrating the principle that the excitation target in the trigger sequence is activated by its adjacent conduction target, thereby modifying the -35box of the TAC promoter.

[0050] Figure 3A schematic diagram showing the relative positions of the excitation target and promoter in the fuse sequence is provided.

[0051] Figure 4 A schematic diagram of a fuse sequence containing an initial conduction target, a conduction target (1), and an excitation target is given.

[0052] Figure 5 This is a map of the pFuse1 plasmid.

[0053] Figure 6 This is a map of the pFuse2 plasmid.

[0054] Figure 7 This is a map of the pFuse3 plasmid.

[0055] Figure 8 The image shows the pACYC-ABE7.10(dCas9) plasmid.

[0056] Figure 9 The colony composition on plates with different incubation times is shown.

[0057] Figure 10 The study showed the colony distribution on different plates under the same incubation period (7 days).

[0058] Figure 11 The fluorescence intensity of colonies on different plates under the same incubation time was analyzed (**** in the figure indicates significant difference, P<0.0001). Detailed Implementation

[0059] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0060] This invention designs a fuse sequence as a gene expression regulatory switch element.

[0061] I. Design Rules for Fuse Sequences

[0062] The overall organization of the trigger sequence (also known as the overall sequence) is as follows:

[0063] 5'-Tac promoter-connector segment-[basic unit]×n-NBB-TGG-3'

[0064] The nucleotide sequence of the Tac promoter is shown in SEQ ID NO.1, while TGTCAA is the -35 box of the Tac promoter.

[0065] The linker sequence is designed as: 5'-NNNNNNNNNNN-3', where N represents any base, that is, the linker has 11 arbitrary bases;

[0066] The sequence design of the basic unit is: 5'-NBBTAABNNNNNNNNNN-3', that is, a basic unit has 17 base pairs, where B represents one of the bases G, T, and C, N represents any base, and n is the number of basic units, n = 1, 2, 3...;

[0067] Let x be the sum of the number of G and C bases in the three bases closest to the 5' end of the basic unit, and y be the sum of the number of G and C bases in all 17 bases of the basic unit. Then x + y = 10. Let z be the sum of the number of G and C bases in the 11 bases of the linker segment. Then x + z = 8.

[0068] Depending on their position within the aforementioned trigger sequence, the trigger sequence initially contains three different sequences of ABE7.10 target sites:

[0069] (1) The initial transduction target (5'-NBB-TAABNNNNNNNNNNNBB-TGG-3') is the part of the overall sequence closest to the 3' end. This target can be recognized by ABE7.10 (dCas9), and the 5th and 6th adenine bases (A) near the 5' end will be modified by ABE7.10 (dCas9) to guanine bases (G).

[0070] (2) The transduction target (5'-NBB-TAABNNNNNNNNNNNBB-TAA-3') is the sequence in the middle of the overall sequence in the initial state. The 3' end of the transduction target does not contain active PAM, therefore it cannot be recognized or modified by ABE7.10 (dCas9) in the initial state. However, because the 3' end of the transduction target overlaps with the editing window of the initial transduction target, after the initial transduction target is edited by ABE7.10 (dCas9), the PAM sequence of the transduction target is converted to TGG, thus transforming the transduction target into a new initial transduction target.

[0071] (3) The activation target (5'-TGTCAA-connector-NBB-TAA-3') is the portion of the overall sequence closest to the Tac promoter. The 3' end of the activation target also lacks active PAM, so it cannot be recognized and modified by ABE7.10 (dCas9) in its initial state. Similarly, since the 3' end of the activation target overlaps with the editing window of the previous transduction target, when the previous transduction target is activated and transformed into a new initial transduction target and modified by ABE7.10 (dCas9), the PAM sequence of the activation target will be converted to TGG, thus becoming recognizable by ABE7.10 (dCas9). This modifies the two outermost adenine bases of the -35box of the Tac promoter, inactivating the Tac promoter.

[0072] The initial transduction target and the transduction target share a common sgRNA, whose sequence is: 5-NBBTAABNNNNNNNNNNNBB-gRNAscaffold-3', which is called the transduction sgRNA;

[0073] The above-mentioned activation target uses sequence 5'-TGTCAA-connector-NBB-gRNAscaffold-3' as the sgRNA, which is called the activation sgRNA.

[0074] It is evident that, for the trigger sequence, in the initial state, only the initial conduction target can be modified by ABE7.10 (dCas9); neither the conduction target nor the excitation target can be modified by ABE7.10 (dCas9). However, after ABE7.10 (dCas9) identifies the target closest to the 3' end (the initial conduction target), it modifies the AA near the 5' end of this target to GG. Since the 5' end of the initial conduction target overlaps with the 3' end of the conduction target, this modification activates the PAM of the conduction target, transforming it into the initial conduction target, which can then be identified by ABE7.10 (dCas9). The AA near the 5' end of this target is then modified to GG. Since the 5' end of this target overlaps with the 3' end of the next conduction target, this modification activates the PAM of the next conduction target. Similarly, modifications near the 5' end of each target site activate the PAM of the next target site, thus sequentially activating each target site from the 3' to the 5' end of the entire sequence, until the PAM of the activation target site near the linker and promoter is activated. After the activation target site is recognized by ABE7.10 (dCas9), the two outermost adenine bases of the -35box of the Tac promoter are modified, inactivating the Tac promoter and thereby regulating gene expression. In the above fusesequence, the sequential activation logic of each target site is similar to a fuse, hence the name "fusesequence".

[0075] Figure 1 This is a schematic diagram illustrating the principle of activating and transforming the adjacent conduction target points of the initial conduction target point closest to the 3' end in the fuse sequence into the initial conduction target point.

[0076] like Figure 1 As shown, AA in the editing window of the initial transduction target is located in the shared region of the two targets. Therefore, AA in the editing window of the initial transduction target is edited as GG by ABE7.10(dCas9), which is equivalent to the inactivated PAM of the transduction target being converted into an active PAM. The transduction target is converted into the initial transduction target, so it can be recognized by ABE7.10(dCas9). Consequently, AA in the editing window of the transduction target is also edited as GG by ABE7.10(dCas9).

[0077] In addition, although Figure 1 It is not shown in the text, but it is obvious that when Figure 1 Once a transduction target is activated and transformed into an initial transduction target, it will form a group with the next transduction target, such as... Figure 1 The initial conduction target point and adjacent conduction target points are shown, so that the next conduction target point will also be activated and transformed into the initial conduction target point. This process is repeated to activate each conduction target point in sequence.

[0078] Figure 2 This is a schematic diagram illustrating the principle that the excitation target in the trigger sequence is activated by its adjacent conduction target, thereby modifying the -35box of the TAC promoter. Figure 3 A schematic diagram showing the relative positions of the excitation target and promoter in the fuse sequence is provided.

[0079] like Figure 2 As shown, AA in the editing window of the transduction target is located in the shared region of the two targets. Therefore, after AA in the editing window of the transduction target is edited to GG by ABE7.10(dCas9), the inactivated PAM of the excitation target is converted into an active PAM, and the excitation target can be recognized by ABE7.10(dCas9). Consequently, the two outermost adenine bases of the -35box of the Tac promoter are edited by ABE7.10(dCas9), and the Tac promoter is inactivated.

[0080] Figure 4 A schematic diagram of a fuse sequence containing an initial conduction target, a conduction target (1), and an excitation target is given. Figure 4 In this process, the initial transduction target and the transduction target share a common region, and the transduction target and the excitation target also share a common region.

[0081] Of course, since the number of basic units n in the fuse sequence can take the value 1, 2, 3..., the fuse sequence can contain only the initial conduction target and the activation target (n=1), or it can contain the initial conduction target, conduction target (1) and activation target (n=2), or it can contain the initial conduction target, conduction target (multiple) and activation target (n>2). In this case, the number of conduction targets is adjustable, m=0, 1, 2, 3...

[0082] It can be observed that in the above trigger sequences, the sequence length varies depending on the value of the number of basic units (n), and the number of target sites that can be edited by ABE7.10 in the initial state also varies. Therefore, by adjusting the number of basic units, the type and / or number of target sites can be adjusted, thus allowing for the pre-setting of the relative time for gene expression regulation. For trigger sequences, the more target sites, the longer the gene expression time. For trigger sequences that simultaneously contain initial, transduction, and activation target sites, the more transduction target sites, the longer the gene expression time. Trigger sequences can serve as regulatory switches for gene expression.

[0083] To illustrate more specifically how trigger sequences regulate the temporal expression of target genes, the following example will be used: a basic unit that conforms to the above-mentioned trigger sequence design rules and corresponding linker segments are selected to construct trigger sequences of different lengths, and the mCherry red fluorescent reporter gene is used as the target gene to characterize the temporal differences in the excitation of trigger sequences of different lengths.

[0084] II. Examples

[0085] (I) Overall Sequence of the Trigger

[0086] Based on the above rules, design the required basic units and connecting segments as follows:

[0087] The nucleotide sequence of the basic unit is shown in SEQ ID NO.2; the nucleotide sequence of the linker segment is shown in SEQ ID NO.3.

[0088] The overall sequence of the triggers is as follows:

[0089] 5'-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTGACG-

[0090] [AGCTAACTGCAGTCACT]×n-NBB-TGG-3'

[0091] Where B represents one of the bases G, T, and C, N represents any base, and n = 1, 2, 3, ...

[0092] In the overall sequence of the aforementioned trigger, the initial transduction target is located in the part of the overall sequence closest to the 3' end, and the nucleotide sequence of the initial transduction target is shown in SEQ ID NO.4; the transduction target is located in the middle of the overall sequence, and the nucleotide sequence of the transduction target is shown in SEQ ID NO.5, m=0,1,2,3……; the excitation target is located in the part of the overall sequence close to the Tac promoter, and the nucleotide sequence of the excitation target is shown in SEQ ID NO.6.

[0093] The initial transduction target and the transduction target share the same transduction sgRNA. The nucleotide sequence of the transduction sgRNA is shown in SEQ ID NO.7 and can be written as: 5'-AGCTAACTGCAGTCACTAGC-gRNAscaffold-3'.

[0094] The sgRNA sequence used to stimulate the target site is the stimulating sgRNA. The nucleotide sequence of the stimulating sgRNA is shown in SEQ ID NO.8, and can also be written as: 5'-TGTCAATCATGCTGACGAGC-gRNAscaffold-3'.

[0095] (II) Design of three different fuse sequences

[0096] Based on the above sequence organization rules, two fuse sequences of different lengths, Fuse2 and Fuse3, are arranged using the designed basic units and connecting segments. The Fuse2 sequence contains an initial conduction target and an excitation target, while the Fuse3 sequence contains an initial conduction target, a conduction target, and an excitation target.

[0097] In addition, for ease of comparison, a sequence containing only one excitation target was designed. Furthermore, to ensure that the individual excitation target could be recognized by ABE7.10, the TAA at the 3' end of the excitation target was modified to TGG. For ease of explanation, this series will be referred to as the Fuse1 sequence.

[0098] Therefore, although the Fuse1 sequence is a specially designed contrast sequence that differs from the fuse sequences Fuse2 and Fuse3, for convenience, the Fuse1, Fuse2, and Fuse3 sequences will be referred to together as three fuse sequences of different lengths in the following description.

[0099] The corresponding sequences are as follows:

[0100] Fuse1 sequence: 5'-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTG ACG-NBB-TGG-3';

[0101] Fuse2 sequence: 5'-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTG ACG-AGCTAACTGCAGTCACT-NBB-TGG-3';

[0102] Fuse3 sequence: 5'-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTG ACG-AGCTAACTGCAGTCACT-AGCTAACTGCAGTCACT-NBB-TGG-3';

[0103] B represents one of the bases G, T, and C, and N represents any base.

[0104] (III) Trigger sequences are used for the temporal regulation of gene expression.

[0105] 3.1 Raw Material Description

[0106] Escherichia coli BL21(DE3) competent cells were purchased from TransGen. DNA polymerase was produced by TransGen. FastPfu Fly DNA Polymerase. The plasmid extraction kit was purchased from AXYGEN Ltd., the PCR product nucleic acid purification kit from Promega Ltd., and the one-step cloning kit from Vazyme Ltd. The Ultra One Step Cloning Kit and the point mutation kit were from TransGen's Fast Mutagenesis System. pCDFDuet-1 and pACYCDuet-1 plasmids were purchased from Novagen. The pCMV-ABE7.10 plasmid, used as a template for the ABE7.10 fragment, was purchased from Addgene. The LB liquid medium formulation was: 10g peptone, 5g yeast extract, 10g NaCl, and 1L deionized water. Tryptone, sodium chloride, and yeast extract were purchased from OXOID. In LB agar plates containing chloramphenicol and streptomycin, the concentration of chloramphenicol was 34ug / ml, and the concentration of streptomycin was 40ug / ml. In LB agar plates containing streptomycin, the concentration of streptomycin was 40ug / ml. Chloramphenicol and streptomycin were purchased from Sangon Biotech.

[0107] 3.2 Sequence synthesis

[0108] The following fragments were synthesized by Beijing Qingke Biotechnology Co., Ltd.: mCherry protein fragment, signaling sgRNA fragment, activation sgRNA fragment, Fuse1 fragment, Fuse2 fragment, and Fuse3 fragment.

[0109] The nucleotide sequence of the mCherry protein fragment is shown in SEQ ID NO. 9.

[0110] The nucleotide sequence of the signaling sgRNA fragment is shown in SEQ ID NO.10. This fragment contains the signaling sgRNA and fragments with low homology to other components to facilitate recombination.

[0111] The nucleotide sequence of the activation sgRNA fragment is shown in SEQ ID NO.11. This fragment contains the activation sgRNA and fragments with low homology to other components to facilitate recombination.

[0112] The nucleotide sequence of the Fuse1 fragment is shown in SEQ ID NO.12. This fragment contains a reserved one-step cloning homologous region (first 21 positions), an antisense strand sequence of the Fuse1 sequence (antiparallel to the Fuse1 sequence), and fragments with low homology to other components to facilitate recombination.

[0113] The nucleotide sequence of the Fuse2 fragment is shown in SEQ ID NO.13. This fragment contains a reserved one-step cloning homologous region (first 21 positions), an antisense strand sequence of the Fuse2 sequence (antiparallel to the Fuse1 sequence), and fragments with low homology to other components to facilitate recombination.

[0114] The nucleotide sequence of the Fuse3 fragment is shown in SEQ ID NO.14. This fragment contains a reserved one-step cloning homologous region (the first 21 positions), an antisense strand sequence of the Fuse3 sequence (antiparallel to the Fuse1 sequence), and segments with low homology to other components to facilitate recombination. In the Fuse3 fragment, positions 73-101 are the tac promoter sequence, and positions 102-146 contain the ribosome binding site (i.e., the Chain-Dalgano sequence). The tac promoter and ribosome binding site ensure that the subsequent mCherry protein sequence can be transcribed and translated normally.

[0115] 3.3 Plasmid Construction

[0116] (1) Construction of pFuse1 plasmid:

[0117] The transducing sgRNA fragment was amplified using primers CD1 and CD2. The Fuse1 fragment was amplified using primers Fuse-1 and Fuse-2. The mCherry fragment was amplified using primers mCherry1 and mCherry2. The plasmid pCDFDuet1-1 was amplified using primers pCDFDuet1-1 and pCDFDuet1-2.

[0118] The nucleotide sequence of the first primer CD1 is shown in SEQ ID NO.15, the nucleotide sequence of the second primer CD2 is shown in SEQ ID NO.16, the nucleotide sequence of the third primer Fuse-1 is shown in SEQ ID NO.17, the nucleotide sequence of the fourth primer Fuse-2 is shown in SEQ ID NO.18, the nucleotide sequence of the fifth primer mCherry1 is shown in SEQ ID NO.19, the nucleotide sequence of the sixth primer mCherry2 is shown in SEQ ID NO.20, the nucleotide sequence of the seventh primer pCDFDuet1-1 is shown in SEQ ID NO.21, and the nucleotide sequence of the eighth primer is shown in SEQ ID NO.22.

[0119] Amplification using Transgen FastPfu Fly DNA Polymerase. The amplification system includes: 1 μL FastPfu Fly DNA Polymerase, 10 μL Fly Buffer, 6 μL 2.5 mM dNTPs, 30 μL deionized water, 1 μL each of primers, and 1 μL template. The PCR program is as follows: (1) 95℃, 3 minutes; (2) 95℃, 30 seconds; (3) 58℃, 30 seconds; (4) 72℃, 2 minutes; repeat steps (2)-(4) for 35 cycles; (5) 72℃, 5 minutes.

[0120] The amplified PCR products were processed using Vazyme's... The Ultra One Step Cloning Kit was used for one-step cloning. The four amplified fragments (sgRNA fragment, Fuse1 fragment, mCherry fragment, and plasmid pCDFDuet-1) were mixed in a 1:1:1:1 ratio, and an equal volume of 2×ClonExpress Mix was added. After ligation in a 50°C water bath for 1 hour, the mixture was cooled on ice for 5 minutes. 10 μL of Transgen-added *E. coli* BL21(DE3) competent cells were then incubated on ice for 30 minutes, followed by heat shock in a 42°C water bath for 75 seconds. 500 μL of LB broth was added, and the mixture was incubated at 37°C with a shaker at 220 rpm for 2 hours. The mixture was then plated onto LB agar plates containing streptomycin, incubated upside down at 37°C for 24 hours, and bacteria were picked and sequenced. After selecting the correct colonies, the pFuse1 plasmid was successfully constructed. The pFuse1 plasmid map is shown below. Figure 5 As shown.

[0121] (2) Construction of pFuse2 plasmid:

[0122] Similar to the construction of the pFuse1 plasmid, the transducing sgRNA fragment was amplified using the first primer CD1 and the second primer CD2, the Fuse2 fragment was amplified using the third primer Fuse-1 and the fourth primer Fuse-2, the mCherry fragment was amplified using the fifth primer mCherry1 and the sixth primer mCherry2, and the plasmid pCDFDuet-1 was amplified using the seventh primer pCDFDuet1-1 and the eighth primer pCDFDuet1-2.

[0123] Amplification using Transgen FastPfu Fly DNA Polymerase. The amplification system includes: 1 μL FastPfu Fly DNA Polymerase, 10 μL Fly Buffer, 6 μL 2.5 mM dNTPs, 30 μL deionized water, 1 μL each of primers, and 1 μL template. The PCR program is as follows: (1) 95℃, 3 minutes; (2) 95℃, 30 seconds; (3) 58℃, 30 seconds; (4) 72℃, 2 minutes; repeat steps (2)-(4) for 35 cycles; (5) 72℃, 5 minutes.

[0124] The amplified PCR products were processed using Vazyme's... The Ultra One Step Cloning Kit was used for one-step cloning. The four fragments (sgRNA fragment, Fuse2 fragment, mCherry fragment, and plasmid pCDFDuet-1) were mixed in a 1:1:1:1 molecular weight ratio, and an equal volume of 2×ClonExpress Mix was added. After ligation in a 50°C water bath for 1 hour, the mixture was cooled on ice for 5 minutes. 10 μL of Transgen-added *E. coli* BL21(DE3) competent cells were then incubated on ice for 30 minutes, followed by heat shock in a 42°C water bath for 75 seconds. 500 μL of LB broth was added, and the mixture was incubated at 37°C with a shaker at 220 rpm for 2 hours. The mixture was then plated onto LB agar plates containing streptomycin, incubated upside down at 37°C for 24 hours, and bacteria were picked and sequenced. After selecting the correct colonies, the pFuse2 plasmid was successfully constructed. The pFuse2 plasmid map is shown below. Figure 6 As shown.

[0125] (3) Construction of pFuse3 plasmid:

[0126] Similar to the construction of the pFuse1 plasmid, the transducing sgRNA fragment was amplified using the first primer CD1 and the second primer CD2, the Fuse3 fragment was amplified using the third primer Fuse-1 and the fourth primer Fuse-2, the mCherry fragment was amplified using the fifth primer mCherry1 and the sixth primer mCherry2, and the plasmid pCDFDuet-1 was amplified using the seventh primer pCDFDuet1-1 and the eighth primer pCDFDuet1-2.

[0127] Amplification using Transgen FastPfu Fly DNA Polymerase. The amplification system includes: 1 μL FastPfu Fly DNA Polymerase, 10 μL Fly Buffer, 6 μL 2.5 mM dNTPs, 30 μL deionized water, 1 μL each of primers, and 1 μL template. The PCR program is as follows: (1) 95℃, 3 minutes; (2) 95℃, 30 seconds; (3) 58℃, 30 seconds; (4) 72℃, 2 minutes; repeat steps (2)-(4) for 35 cycles; (5) 72℃, 5 minutes.

[0128] The amplified PCR products were processed using Vazyme's... The Ultra One Step Cloning Kit was used for one-step cloning. The four amplified fragments (sgRNA fragment, Fuse3 fragment, mCherry fragment, and plasmid pCDFDuet-1) were mixed at a molecular weight ratio of 1:1:1:1, and an equal volume of 2×ClonExpress Mix was added. After ligation in a 50°C water bath for 1 hour, the mixture was cooled on ice for 5 minutes. 10 μL of Transgen-added *E. coli* BL21(DE3) competent cells were then incubated on ice for 30 minutes, followed by heat shock in a 42°C water bath for 75 seconds. 500 μL of LB broth was added, and the mixture was incubated at 37°C with a shaker at 220 rpm for 2 hours. The mixture was then plated onto LB agar plates containing streptomycin, incubated upside down at 37°C for 24 hours, and bacteria were picked and sequenced. After selecting the correct colonies, the pFuse3 plasmid was successfully constructed. The pFuse3 plasmid map is shown below. Figure 7 As shown.

[0129] (4) Construction of pJF plasmid:

[0130] The excitation sgRNA fragment was amplified using primers JF1 (ninth primer) and JF2 (tenth primer), and plasmid pACYCDuet-1 was amplified using primers pACYCDuet1-1 (eleventh primer) and pACYCDuet1-2 (twelfth primer).

[0131] The nucleotide sequence of the ninth primer JF1 is shown in SEQ ID NO.23, the nucleotide sequence of the tenth primer JF2 is shown in SEQ ID NO.24, the nucleotide sequence of the eleventh primer pACYCDuet1-1 is shown in SEQ ID NO.25, and the nucleotide sequence of the twelfth primer pACYCDuet1-2 is shown in SEQ ID NO.26.

[0132] Amplification using Transgen FastPfu Fly DNA Polymerase. The amplification system includes: 1 μL FastPfu Fly DNA Polymerase, 10 μL Fly Buffer, 6 μL 2.5 mM dNTPs, 30 μL deionized water, 1 μL each of primers, and 1 μL template. The PCR program is as follows: (1) 95℃, 3 minutes; (2) 95℃, 30 seconds; (3) 58℃, 30 seconds; (4) 72℃, 2 minutes; repeat steps (2)-(4) for 35 cycles; (5) 72℃, 5 minutes.

[0133] The amplified PCR products were processed using Vazyme's... The Ultra One Step Cloning Kit was used for one-step cloning. The two amplified fragments (excitation sgRNA fragment and plasmid pACYCDuet-1) were mixed at a molecular weight ratio of 1:1, and an equal volume of 2×ClonExpress Mix was added. After ligation in a 50°C water bath for 1 hour, the mixture was cooled on ice for 5 minutes. 10 μL of Transgen's E. coli BL21(DE3) competent cells were added, and the mixture was incubated on ice for 30 minutes. After heat shock in a 42°C water bath for 75 seconds, 500 μL of LB liquid medium was added. The mixture was cultured in a shaker at 37°C and 220 rpm for 2 hours. The mixture was then plated onto LB agar plates containing streptomycin and incubated upside down at 37°C for 24 hours. After picking and sequencing, the pJF plasmid was constructed after the correct colonies were picked.

[0134] (5) Construction of pACYC-ABE7.10 plasmid:

[0135] The ABE7.10 plasmid was amplified using primers ABE7.10-1 (13th primer) and ABE7.10-2 (14th primer), and plasmid pJF was amplified using primers pJF-1 (15th primer) and pJF-2 (16th primer).

[0136] The nucleotide sequence of primer ABE7.10-1 is shown in SEQ ID NO.27, the nucleotide sequence of primer ABE7.10-2 is shown in SEQ ID NO.28, the nucleotide sequence of primer pJF-1 is shown in SEQ ID NO.29, and the nucleotide sequence of primer pJF-2 is shown in SEQ ID NO.30.

[0137] Amplification using Transgen FastPfu Fly DNA Polymerase. The amplification system includes: 1 μL FastPfu Fly DNA Polymerase, 10 μL Fly Buffer, 6 μL 2.5 mM dNTPs, 30 μL deionized water, 1 μL each of primers, and 1 μL template. The PCR program is as follows: (1) 95℃, 3 minutes; (2) 95℃, 30 seconds; (3) 58℃, 30 seconds; (4) 72℃, 2 minutes; repeat steps (2)-(4) for 35 cycles; (5) 72℃, 5 minutes.

[0138] The amplified PCR products were processed using Vazyme's... The Ultra One Step Cloning Kit was used for one-step cloning. The two amplified fragments (ABE7.10 plasmid and pJF plasmid) were mixed at a molecular weight ratio of 1:1, and an equal volume of 2×ClonExpress Mix was added. After ligation in a 50°C water bath for 1 hour, the mixture was cooled on ice for 5 minutes. 10 μL of Transgen's E. coli BL21(DE3) competent cells were added, and the mixture was incubated on ice for 30 minutes. After heat shock in a 42°C water bath for 75 seconds, 500 μL of LB liquid medium was added. The mixture was incubated at 37°C on a shaker at 220 rpm for 2 hours. The mixture was then plated onto LB agar plates containing streptomycin and incubated upside down at 37°C for 24 hours. After picking and sequencing, the pACYC-ABE7.10 plasmid was successfully constructed after the correct colonies were picked.

[0139] (6) Construction of pACYC-ABE7.10(dCas9) plasmid

[0140] The pACYC-ABE7.10 plasmid was amplified using primers H840A-1 (17th primer) and H840A-2 (18th primer). The nucleotide sequence of primer H840A-1 is shown in SEQ ID NO.31, and the nucleotide sequence of primer H840A-2 is shown in SEQ ID NO.32.

[0141] The amplification system included: 1 μL FastPfu Fly DNA Polymerase, 10 μL Fly Buffer, 6 μL 2.5 mM dNTPs, 30 μL deionized water, 1 μL each of primers, and 1 μL template. The PCR program was as follows: (1) 95℃, 3 minutes; (2) 95℃, 30 seconds; (3) 58℃, 30 seconds; (4) 72℃, 2 minutes; repeat steps (2)-(4) for 25 cycles; (5) 72℃, 5 minutes.

[0142] Add 1 μL of DMT enzyme from Transgen's Fast Mutagenesis System to the product. Incubate the mixture in a 37°C water bath for 1 hour. Then, take 10 μL of E. coli BL21(DE3) competent cells containing Transgen, incubate on ice for 30 minutes, heat shock in a 42°C water bath for 75 seconds, add 500 μL of LB liquid medium, and incubate at 37°C with a shaker at 220 rpm for 2 hours. Spread the mixture onto LB agar plates containing streptomycin, incubate upside down at 37°C for 24 hours, pick bacteria, and sequence. After selecting the correct colonies, the pACYC-ABE7.10(dCas9) plasmid construction is complete. This process can also be referred to as the H840A point mutation of pACYC-ABE7.10(dCas9). The pACYC-ABE7.10(dCas9) plasmid map is shown below. Figure 8 As shown.

[0143] 3.4 Transformation of E. coli with plasmid DNA

[0144] 200 ng pFuse1 plasmid and 250 ng pACYC-ABE7.10(dCas9) plasmid were added to Transgen's E. coli BL21(DE3) competent cells. The cells were incubated on ice for 30 minutes, then heat-shocked at 42°C for 75 seconds in a water bath. 500 μL of LB liquid medium was added, and the cells were cultured at 37°C and 220 rpm for two hours. The culture was then spread on LB agar plates containing chloramphenicol and streptomycin antibiotics. After incubation at 37°C with the plates inverted for a period of time, the plates were observed.

[0145] 200 ng pFuse2 plasmid and 250 ng pACYC-ABE7.10(dCas9) plasmid were added to Transgen's E. coli BL21(DE3) competent cells. The cells were incubated on ice for 30 minutes, then heat-shocked at 42°C for 75 seconds in a water bath. 500 μL of LB liquid medium was added, and the cells were cultured at 37°C and 220 rpm for two hours. The culture was then spread on LB agar plates containing chloramphenicol and streptomycin antibiotics. After incubation at 37°C with the plates inverted for a period of time, the plates were observed.

[0146] 200 ng pFuse3 plasmid and 250 ng pACYC-ABE7.10(dCas9) plasmid were added to Transgen's E. coli BL21(DE3) competent cells. The cells were incubated on ice for 30 minutes, then heat-shocked at 42°C for 75 seconds in a water bath. 500 μL of LB liquid medium was added, and the cells were cultured at 37°C and 220 rpm for two hours. The culture was then spread on LB agar plates containing chloramphenicol and streptomycin antibiotics. After incubation at 37°C with the plates inverted for a period of time, the plates were observed.

[0147] Control group: 200 ng pCDFDuet-1 plasmid and 250 ng pACYC-ABE7.10(dCas9) plasmid were added to Transgen's E. coli BL21(DE3) competent cells, incubated on ice for 30 minutes, heat-shocked at 42°C for 75 seconds in a water bath, 500 μL LB liquid medium were added, and the cells were cultured at 37°C and 220 rpm for two hours. The cells were then plated on LB medium plates containing chloramphenicol and streptomycin antibiotics and cultured upside down at 37°C for a period of time before the plates were observed.

[0148] 3.5 Results

[0149] Observe and record the growth of colonies on plate 3 at incubation times of 2, 3, 4, 5, and 7 days. Figure 9 As shown. Figure 9 The development process of chimeric colonies expressing red fluorescent protein on plate 3 was recorded. The dark areas of the colonies were red due to mCherry expression, and the light areas were milky white due to control colonies (E. coli).

[0150] from Figure 9 As can be seen, when the incubation time is short, the colonies are predominantly red, reflecting mCherry expression. However, as the incubation time increases, the newly grown areas around the colonies become milky white, indicating the absence of mCherry expression. After a period of time, the milky white bacteria completely surround the outer edge of the colony. This is because the gene controlled by the regulatory element switch becomes inactive after a certain period; that is, the mCherry gene stops expressing itself. Therefore, two phenotypes exist in colonies grown on the plate: one expressing mCherry, appearing red, and the other not expressing mCherry, appearing milky white. Because the cells at the periphery of the colony are newer, they tend to exhibit the white phenotype of not expressing mCherry, indicating a delayed gene expression. Conversely, the cells in the center of the colony, which grew earlier, tend to exhibit the red phenotype of expressing mCherry, indicating an earlier gene expression.

[0151] Under the same incubation period (7 days), plate 1, plate 2, and plate 3 were observed, as follows: Figure 10As shown, the colony appearances of the three plates differ: the colonies on plate 3 are generally more reddish, those on plate 1 are generally more whitish, and the color of the colonies on plate 2 is somewhere in between. This indicates that, under the same incubation time (7 days), the colonies carrying pFuse3 express mCherry for the longest time, and the area expressing mCherry on plate 3 is more extensive; the colonies carrying pFuse1 express mCherry for the shortest time, and the area expressing mCherry on plate 1 is less extensive; the colonies carrying pFuse2 express mCherry for a time between the two, and the area expressing mCherry on plate 2 is also somewhere in between. This is because pFuse3 has the most target sites, pFuse1 has the fewest, and pFuse2 has a target site in between. Different numbers of target sites result in different activation times, thus affecting the time it takes to reach and activate the excitation target, thereby modifying the two outermost adenine bases of the -35box of the Tac promoter to inactivate it. Therefore, the duration of mCherry expression varies. This indicates that regulatory elements containing three different lengths of trigger sequences (pFuse1, pFuse2, and pFuse3) have varying numbers of target sites, resulting in different durations of mCherry expression. In other words, the temporal regulation of mCherry gene expression can be achieved by adjusting the number of target sites in the trigger sequence. For example, increasing the number of transducing target sites in the trigger sequence prolongs the expression of mCherry in colonies carrying that trigger sequence.

[0152] To characterize the differences in total mCherry expression in colonies with different fuse lengths, fluorescence characterization was performed on the three types of plates and the control plate. Colonies from each plate grown for 7 days were washed completely with 1×PBS buffer and diluted to OD600 = 1.0. In standard 96-well plates with clear bottoms, colonies of BL21(DE3) that do not express mCherry on plate 4 (control group) were used as a control. Fluorescence intensity was measured using a SpectraMax M5 microplate reader from Molecular Divices. The excitation wavelength was 587 nm, the emission wavelength was 610 nm, the sample volume was 100 μL, and the number of replicates was 3. Figure 11 The fluorescence intensity data shown.

[0153] Depend on Figure 11It can be seen that the fluorescence intensity of colonies carrying pFuse3 is higher than that of colonies carrying pFuse2 and pFuse1, indicating that the longer the trigger sequence, the more the reporter gene (mCherry red fluorescent gene) is expressed, and the slower the reporter gene inactivation rate. In the inactive state, the tac promoter of the gene regulatory element is not modified, and the mCherry fluorescent protein is expressed normally, exhibiting red fluorescence in E. coli; in the activated state, the tac promoter of the gene regulatory element is modified, mCherry expression is inactivated, and the red fluorescence disappears. This shows that, under the same ABE7.10 (dCas9), activation sgRNA, and transduction sgRNA concentration levels, the shorter the trigger sequence length, the shorter the time required for mCherry expression to stop. Therefore, the timing of gene expression can be controlled by adjusting the length of the trigger sequence. This proves that the timing of gene expression can be controlled by pre-programming the length of the trigger sequence of the regulatory element.

[0154] Based on the same principle, trigger sequences conforming to the design rules in the embodiments of this invention can all achieve the aforementioned function of temporal regulation of gene expression. The basic unit and linker segment are not limited to one type in the examples; under the design rules of this invention, they can be adjusted as needed so that dCas9 binds to the target site matched by the sgRNA via sgRNA. Furthermore, the number of target sites (i.e., the length of the trigger sequence) can also be adjusted according to actual requirements.

[0155] Furthermore, the tac promoter used in this invention is a universal promoter in E. coli, which can efficiently start its downstream genes. Therefore, it is also possible to replace mCherry in the example with other reporter genes. Similarly, it is also feasible to select functional genes or regulatory factors (such as functional genes or regulatory factors of natural product anabolic pathways, antibiotic anabolic pathways, and environmental pollutant degradation pathways) as target genes.

[0156] Therefore, the applications of the above-mentioned gene expression regulatory elements or systems include, but are not limited to: pre-programming for the temporal sequential expression of genes synthesizing different metabolite pathways in engineered strains; gene expression control for engineered strains synthesizing natural products and antibiotics; and gene expression control for engineered bacteria synthesizing environmental pollutant treatment. sequence list <110> Zhejiang University <120> Gene temporal expression regulation systems, methods, and applications based on single-base editing <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <400> 1 cattatacga gccgatgatt aattgtcaa 29 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 agctaactgc agtcact 17 <210> 3 <211> 11 <212> DNA <213> Artificial Sequence <400> 3 tcatgctgac g 11 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 agctaactgc agtcactagc tgg 23 <210> 5 <211> twenty three <212> DNA <213> Artificial Sequence <400> 5 agctaactgc agtcactagc taa 23 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 tgtcaatcat gctgacgagc taa 23 <210> 7 <211> 104 <212> DNA <213> Artificial Sequence <400> 7 agctaactgc agtcactagc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttt 104 <210> 8 <211> 104 <212> DNA <213> Artificial Sequence <400> 8 tgtcaatcat gctgacgagc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttt 104 <210> 9 <211> 711 <212> DNA <213> MCherry fluorescent protein <400> 9 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711 <210> 10 <211> 259 <212> DNA <213> Artificial Sequence <400> 10 ttgacagcta gctcagtcct aggtataata ctagtagcta actgcagtca ctagcgtttt 60 ttgacagcta gctcagtcct aggtataata ctagtagcta actgcagtca ctagcgtttt 60 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 120 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 120 cgagtcggtg cttttttttg gcggccgcat aatgcttaag tcgaacagaa agtaatcgta 180 ttgtacacgg ccgcgggatc tcgacgctct cccttatgcg actccgcaag gaatggtaat 240 gggtcgcgga tccgaattc 259 <210> 11 <211> 205 <212> DNA <213> Artificial Sequence <400> 11 ttgacagcta gctcagtcct aggtataata ctagttgtca atcatgctga cgagcgtttt 60 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 120 cgagtcggtg cttttttttg gcggccgcat aatgcttaag tcgaacagaa agtaatcgta 180 ttgtacacgg ccgcataatc gaaat 205 <210> 12 <211> 112 <212> DNA <213> Artificial Sequence <400> 12 atgggtcgcg gatccgaatt cccagctcgt cagcatgatt gacaattaat catcggctcg 60 tataatgttt ccctctagaa ataatcctct agaaataata ggaggaaaac tt 112 <210> 13 <211> 129 <212> DNA <213> Artificial Sequence <400> 13 atgggtcgcg gatccgaatt cccagctagt gactgcagtt agctcgtcag catgattgac 60 aattaatcat cggctcgtat aatgtttccc tctagaaata atcctctaga aataatagga 120 ggaaaactt 129 <210> 14 <211> 146 <212> DNA <213> Artificial Sequence <400> 14 atgggtcgcg gatccgaatt cccagctagt gactgcagtt agctagtgac tgcagttagc 60 tcgtcagcat gattgacaat taatcatcgg ctcgtataat gtttccctct agaaataatc 120 ctctagaaat aataggagga aaactt 146 <210> 15 <211> 45 <212> DNA <213> Artificial Sequence <400> 15 ctgcattagg ttgacagcta gctcagtcct aggtataata ctagt 45 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <400> 16 cactagctgg gaattcggat ccgcgaccca ttacc 35 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <400> 17 atccgaattc ccagctagtg actgcagtta gctcg 35 <210> 18 <211> 55 <212> DNA <213> Artificial Sequence <400> 18 tgctcaccat aagttttcct cctattattt ctagaggatt atttctagag ggaaa 55 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <400> 19 aggaaaactt atggtgagca agggcgagga g 31 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <400> 20 tcgggctttg ttacttgtac agctcgtcca tgccg 35 <210> twenty one <211> 36 <212> DNA <213> Artificial Sequence <400> twenty one tagctgtcaa cctaatgcag gagtcgcata aggggag 36 <210> twenty two <211> 35 <212> DNA <213> Artificial Sequence <400> twenty two gtacaagtaa caaagcccga aaggaagctg agttg 35 <210> twenty three <211> 45 <212> DNA <213> Artificial Sequence <400> twenty three ctgcattagg ttgacagcta gctcagtcct aggtataata ctagt 45 <210> twenty four <211> 36 <212> DNA <213> Artificial Sequence <400> twenty four agtcgtatta atttcgatta tgcggccgtg tacaat 36 <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <400> 25 taatcgaaat taatacgact cactataggg gaattgtgag cg 42 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <400> 26 tagctgtcaa cctaatgcag gagtcgcata aggggag 36 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <400> 27 agatatacat atgtccgaag tcgagttttc ccatga 36 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <400> 28 tggcagcagc ctaggttaag tcacccccaa gctgtg 36 <210> 29 <211> 28 <212> DNA <213> Artificial Sequence <400> 29 cttaacctag gctgctgcca ccgctgag 28 <210> 30 <211> 60 <212> DNA <213> Artificial Sequence <400> 30 cttcggacat atgtatatct ccttcttata cttaactaat atactaagat ggggaattgt 60 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <400> 31 gattacgacg tcgatgcgat tgtaccccaa tcctt 35 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <400> 32 gtacaatcgc atcgacgtcg taatcagata aacggt 36

Claims

1. A gene expression regulatory switching element, characterized in that, The overall sequence of the gene expression regulatory switching element is as follows: 5'-Tac promoter-connector segment-[basic unit]×n-NBB-TGG-3' in, The sequence of the basic unit is: 5'-NBBTAABNNNNNNNNNN-3'; The sequence of the connecting segment is: 5'-NNNNNNNNNNN-3'; The nucleotide sequence of the Tac promoter is shown in SEQ ID NO. 1, wherein TGTCAA is the -35box of the Tac promoter; B represents one of the bases G, T, and C; N represents any base; and n is the number of basic units, n = 1, 2, 3, ... Let x be the sum of the number of G and C bases in the three bases closest to the 5' end of the basic unit, and y be the sum of the number of G and C bases in all 17 bases of the basic unit, then x + y = 10; let z be the sum of the number of G and C bases in all 11 bases of the linker segment, then x + z = 8.

2. The gene expression regulation switching element as described in claim 1, characterized in that, The gene expression regulatory switch element initially has one initial transduction target site, m transduction target sites, and one activation target site, where m = 0, 1, 2, 3...; where, The initial transduction target point is located in the portion of the overall sequence closest to the 3' end, and the sequence of the initial transduction target point is: 5'-NBB-TAABNNNNNNNNNNNBB-TGG-3'; The transduction target is located in the middle of the overall sequence, and the sequence of the transduction target is: 5'-NBB-TAABNNNNNNNNNNNBB-TAA-3'; The excitation target is located in the portion of the overall sequence near the Tac promoter, and the sequence of the excitation target is: 5'-TGTCAA-connector segment-NBB-TAA-3'; The initial transduction target and the transduction target share a single sgRNA, called the transduction sgRNA, and the sequence of the transduction sgRNA is: 5-NBBTAABNNNNNNNNNNNBB-gRNAscaffold-3'; The sgRNA used to stimulate the target site is called the stimulating sgRNA, and the sequence of the stimulating sgRNA is 5'-TGTCAA-connector-NBB-gRNAscaffold-3'.

3. The gene expression regulation switching element as described in claim 1, characterized in that, The nucleotide sequence of the Tac promoter is shown in SEQ ID NO. 1, the nucleotide sequence of the basic unit is shown in SEQ ID NO. 2, and the nucleotide sequence of the linker is shown in SEQ ID NO. 3; the overall sequence of the gene expression regulatory switch element is as follows: 5'-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTGACG-[AGCTAACTGCAGTCACT]×n-NBB-TGG-3' Where B represents one of the bases G, T, and C, N represents any base, and n = 1, 2, 3, ...

4. The gene expression regulation switching element as described in claim 3, characterized in that, The gene expression regulatory switch element initially has one initial transduction target site, m transduction target sites, and one activation target site, where m = 0, 1, 2, 3...; where, The initial transduction target is located in the portion of the overall sequence closest to the 3' end, and the nucleotide sequence of the initial transduction target is shown in SEQ ID NO. 4; the transduction target is located in the middle of the overall sequence, and the nucleotide sequence of the transduction target is shown in SEQ ID NO. 5, m=0,1,2,3...; the activation target is located in the portion of the overall sequence close to the Tac promoter, and the nucleotide sequence of the activation target is shown in SEQ ID NO. 6; The initial transduction target and the transduction target share a single sgRNA, referred to as the transduction sgRNA, the nucleotide sequence of which is shown in SEQ ID NO. 7; the sgRNA sequence used for the activation target is the activation sgRNA, the nucleotide sequence of which is shown in SEQ ID NO.

8.

5. A gene expression regulation system, comprising: pFuse plasmid and pACYC-ABE7.10-dCas9 plasmid, among which, The pFuse plasmid is obtained by the following method: cloning a gene expression regulatory switch element of a predetermined length as described in any one of claims 1 to 4 into the pCDFDuet-1 plasmid to obtain a trigger plasmid; then cloning the target gene and the signaling sgRNA into the trigger plasmid to obtain the pFuse plasmid; The pACYC-ABE7.10-dCas9 plasmid was obtained by the following method: the 840th residue of ABE7.10 was point-mutated from histidine to alanine to obtain ABE7.10-dCas9; then the ABE7.10-dCas9 and the activation sgRNA were cloned into pACYCDuet-1 to obtain the pACYC-ABE7.10-dCas9 plasmid.

6. The gene expression regulation system as described in claim 5, characterized in that, The target gene includes functional genes or regulatory factors.

7. A method for regulating gene expression, comprising: (1) Design and synthesize a gene expression regulatory switch element of a predetermined length as described in any one of claims 1 to 4, clone the gene expression regulatory switch element into the pCDFDuet-1 plasmid to construct a fuse plasmid, clone the target gene and the signaling sgRNA into the fuse plasmid to obtain the pFuse plasmid; (2) The 840th residue of ABE7.10 was point-mutated from histidine to alanine to obtain ABE7.10-dCas9; then the ABE7.10-dCas9 and the activation sgRNA were cloned into pACYCDuet-1 to obtain the pACYC-ABE7.10-dCas9 plasmid. (3) The pFuse plasmid and pACYC-ABE7.10-dCas9 plasmid were simultaneously transformed into recipient cells to regulate the expression of the target gene.

8. The method for regulating gene expression as described in claim 7, characterized in that, In step (3), the pFuse plasmid and the pACYC-ABE7.10-dCas9 plasmid are simultaneously transformed into E. coli competent cells to regulate the expression of the target gene.

9. The method for regulating gene expression as described in claim 7, characterized in that, The target gene includes functional genes or regulatory factors.

10. The application of the gene expression regulation system as described in claim 5, comprising: Pre-programming for the sequential expression of genes synthesizing different metabolites in engineered strains; Gene expression control of engineered strains used for synthesizing natural products and antibiotics; gene expression control of engineered bacteria used for synthesizing environmental pollutant treatment.

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