CN117106773A - Gene time sequence expression regulation and control system and method based on single base editing and application - Google Patents

Abstract

The invention discloses a gene time sequence expression regulation system and method based on single base editing and application thereof. The invention constructs a series DNA sequence identified by ABE7.10 as a gene expression regulation and control switch element, carries out single base editing on a plurality of adenine targets on the DNA sequences which are mutually influenced and arranged in series by the ABE7.10, and finally realizes the activation or inhibition of the delayed regulation and control target gene expression by modifying the-35 box sequence of the promoter to close or open the promoter overlapped by the last target. The invention can control the time of one or more genes by changing the number (length) of the DNA sequences of the detonating cord, and the more adenine-containing targets are, the longer the time required for the regulation of the target genes is, thereby realizing the regulation of the preset gene expression timing.

Description

Gene temporal expression regulation system and method based on single base editing and its application

Technical field

The invention belongs to the technical field of molecular biology, and specifically relates to a gene temporal expression control system, method and application based on single base editing.

Background technique

Gene dynamic expression regulatory elements are an important field of synthetic biology research. Gene expression regulation can be achieved by changing cis-acting elements through genome editing.

The single base editing system is based on Cas9 (dCas9) and Cas9 nickase (nCas9) without endonuclease activity. Two systems with single base editing functions were obtained by David Liu's laboratory using phage directed evolution. Fusion proteins: cytosine base editor (CBE) and adenine base editor (ABE). In the single base editing system, dCas9 or nCas9 does not have the endonuclease activity to cut the DNA double strands, but retains the DNA binding activity. Without breaking the DNA double strands and providing a DNA template, sgRNA can complement the target site by complementing the target site. Pairing guides the dCas9 or nCas9 in the fusion deaminase protein to bind to the target site and directly edit the target site accurately, achieving a certain activity window from cytosine (C) to thymine (T) or guanine ( G) Single base conversion to adenine (A). Among them, CBE can edit cytosine (C) in the specific target site of the DNA double strand into thymine (T), converting C·G into T·A; ABE can edit the adenosine (C) in the specific target site of the DNA double strand. Purine (A) is edited into guanine (G), converting A·T into G·C.

ABE is a fusion protein of Escherichia coli adenine deaminase and Streptococcus pyogenes nCas9 protein, which was evolved through phage-assisted evolution (Gaudelli N M, Komor A C, Rees H A, et al., Nature, 2017, 551, 464-471) . Taking ABE7.10 as an example, the base editor ABE7.10, which is directed evolution from the fusion protein of spdCas9 and TadA, adds an inactive E. coli deaminase (TadA*) to the N-terminus of nCas9 (D10A). and an active E. coli deaminase (TadA) and obtained through amino acid optimization. The three proteins are connected by a flexible linker. Its function is to recognize the target with an NGG sequence at the 3' end through sgRNA. site, after binding, TadA binds to the single-stranded region (homologous sequence of the original spacer) to deaminate adenine (A) in positions 4-7 of the original spacer to hypoxanthine (I), and hypoxanthine and Cytosine pairs (C), so after subsequent DNA replication is completed, the original position of adenine (A) will be replaced by guanine (G) paired with cytosine (C), and the other strand of the duplex is The HNH domain of nCas9(D10A) cleaves, 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 region. These 4-7 bases are called the edit window.

The recognition DNA sequence site of ABE consists of two parts: a 20 bp protospacer and a protospacer adjacent site NGG (protospacer adjacent motif, hereinafter referred to as PAM) immediately adjacent to the 3' end of the protospacer. Its overall target The sequence is 5'-(N*20)(NGG)-3', in which the original spacer can be directly used as the design template for the spacer of sgRNA. The spacer can be 2bp (inclusive) or less from the original spacer. Differences may reduce the binding efficiency. If the PAM or NGG sequence is wrong (GG is replaced with other bases), ABE will be almost unable to bind.

Since the single base editing system can achieve efficient single base replacement without introducing double-strand breaks, it avoids the uncontrollable introduction of traditional CRISPR/Cas9 when performing nonhomologous end-joining (NHEJ). Insertion or deletion mutations (Indels), therefore, the emergence of single base editing technology has promoted the effectiveness and scope of use of point mutation gene editing. However, there are no relevant literature and patent reports on the use of ABE technology to achieve temporal regulation of gene expression.

Contents of the invention

In view of this, the purpose of the present invention is to provide a gene temporal expression control system, method and application based on single base editing. The present invention constructs a gene expression control switch element, uses ABE7.10 to perform single base editing on several adenine target points on the DNA sequences that interact with each other and are arranged in series, and finally realizes closing or opening by modifying the -35box sequence of the promoter. The last target overlaps the promoter to achieve the purpose of delaying the activation or suppression of the expression of the target gene.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The invention discloses a gene expression control switch element. The overall organization mode (or overall sequence) of the element is as follows:

5’-Tac promoter-adapter-[basic unit]×n-NBB-TGG-3’

in,

The sequence of the basic unit is: 5’-NBBTAABNNNNNNNNNN-3’;

The sequence of the linker is: 5’-NNNNNNNNNNN-3’;

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

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

Suppose the sum of the number of G and C in the three bases closest to the 5' end of the basic unit is x, and the sum of the number of G and C in all 17 bases of the basic unit is y, then x+y= 10; Suppose the number of G and C in all 11 bases of the adapter segment is z, then x+z=8.

In some specific examples of the present invention, the gene expression regulation switch element has 1 initial conduction target point, m conduction target points and 1 excitation target point in the initial state, m=0,1,2,3... …;in,

The initial conduction target is located in the part closest to the 3' end of the overall sequence, and the sequence of the initial conduction target is: 5'-NBB-TAABNNNNNNNNNNBB-TGG-3';

The conduction target is located in the middle of the overall sequence, and the sequence of the conduction target is: 5’-NBB-TAABNNNNNNNNNNNBB-TAA-3’;

The excitation target point is located in the part of the overall sequence close to the Tac promoter, and the sequence of the excitation target point is: 5’-TGTCAA-adapter segment-NBB-TAA-3’;

The initial conduction target and the conduction target share one sgRNA, which is called conduction sgRNA. The sequence of the conduction sgRNA is: 5-NBBTAABNNNNNNNNNNNBB-gRNAscaffold-3’;

The sgRNA used to excite the target is called excitation sgRNA, and the sequence of the excitation sgRNA is 5’-TGTCAA-adapter-NBB-gRNAscaffold-3’.

In some specific examples 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 adapter section The nucleotide sequence is shown in SEQ ID NO.3; the overall organization (or overall sequence) of the gene expression control switch element is as follows:

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

Among them, B represents a base among G, T, and C, N represents any base, n=1, 2, 3...

In some specific examples of the present invention, the gene expression regulation switch element has 1 initial conduction target point, m conduction target points and 1 excitation target point in the initial state, m=0,1,2,3... …;in,

The initial conduction target point is located in the part closest to the 3' end of the overall sequence. The nucleotide sequence of the initial conduction target point is shown in SEQ ID NO. 4; the conduction target point is located in the middle of the overall sequence. , the nucleotide sequence of the conductive target point is shown in SEQ ID NO.5, m=0,1,2,3...; the excitation target point is located in the part of the overall sequence close to the Tac promoter, so The nucleotide sequence of the excitation target is shown in SEQ ID NO. 6;

The initial conduction target and the conduction target share one sgRNA, which is called conduction sgRNA. The nucleotide sequence of the conduction sgRNA is shown in SEQ ID NO.7; the sgRNA sequence used for the excitation target is excitation sgRNA. The nucleotide sequence of the stimulating sgRNA is shown in SEQ ID NO. 8.

In the gene expression control switch element of the present invention, in the initial state, only the initial conduction target can be recognized and edited by ABE7.10. However, after the initial conduction target is modified by ABE7.10, due to the initial conduction target The 5' end of the dot overlaps with the 3' end of the conduction target. The above modification causes the PAM of the conduction target to be activated. The conduction target is converted into the initial conduction target and then modified, and activates the next conduction target to By analogy, each target point of the entire sequence from the 3' end to the 5' end will be activated sequentially, and finally the two outermost adenine bases of the -35box of the Tac promoter are modified to inactivate the Tac promoter, thereby achieving regulation. Gene expression purposes. The logic of sequential activation of each target is similar to that of a fuse. Therefore, the gene expression regulation switch element is named "Fuse sequence".

The invention also discloses a gene expression control system, including: pFuse plasmid and pACYC-ABE7.10 (dCas9) plasmid, wherein,

The pFuse plasmid is obtained by the following method: clone the gene expression control switch element of a preset length into the pCDFDuet-1 plasmid to obtain a fuse plasmid; then clone the target gene and the conductive sgRNA into the fuse plasmid to obtain pFuse plasmid;

The pACYC-ABE7.10 (dCas9) plasmid is obtained by the following method: point mutation of the 840th residue histidine of ABE7.10 to alanine to obtain ABE7.10 (dCas9); and then the ABE7. 10 (dCas9) and the excitation sgRNA were cloned into pACYCDuet-1 to obtain pACYC-ABE7.10 (dCas9) plasmid.

In some specific examples of the present invention, the target gene includes a functional gene or regulatory factor.

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

The invention also discloses a method for regulating gene expression, including:

(1) Design and synthesize the above-mentioned gene expression control switch element of a predetermined length, clone the gene expression control switch element into the pCDFDuet-1 plasmid, thereby constructing a fuse plasmid, and clone the target gene and the conductive sgRNA into the pCDFDuet-1 plasmid. Describe the fuse plasmid to obtain pFuse plasmid;

(2) Point-mute the 840th residue histidine of ABE7.10 to alanine to obtain ABE7.10 (dCas9); then clone the ABE7.10 (dCas9) and the stimulating sgRNA into pACYCDuet- 1. Obtain pACYC-ABE7.10(dCas9) plasmid.

(3) Transform the pFuse plasmid and pACYC-ABE7.10 (dCas9) plasmid into recipient cells at the same time to regulate the expression of the target gene.

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

In some specific examples of the present invention, the target gene includes a functional gene or regulatory factor.

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

The invention also discloses the application of the above-mentioned gene expression control switch element or gene expression control system, including but not limited to: pre-programming for temporal sequential expression of synthetic genes of different metabolite pathways of engineering strains; for synthesis Gene expression control of engineered strains of natural products and antibiotics; gene expression control of engineered strains used to synthesize environmental pollutants.

In the present invention, ABE7.10(dCas9) is also referred to as ABE7.10-dCas9, and pACYC-ABE7.10(dCas9) plasmid is also referred to as pACYC-ABE7.10-dCas9 plasmid.

Compared with the existing technology, the present invention has the following beneficial technical effects:

The present invention constructs a tandem DNA sequence (named Fusesequence, Chinese name "fuse sequence") recognized by the adenine base editing enzyme ABE7.10 as a gene expression control switch element, which interacts with each other and is arranged in tandem through ABE7.10 Single base editing is performed on several adenine target sites on the DNA sequence. Finally, by modifying the -35box sequence of the promoter, the promoter overlapping the last target site is closed or opened to achieve delayed regulation of the activation of gene expression. or containment purposes.

The present invention can control the time when one or more gene expression occurs by changing the number of basic units (or the sequence length of the element) in the gene expression control switch element. The more adenine-containing targets there are, the longer it takes for target gene regulation to occur, allowing for preset regulation of gene expression timing.

The regulatory element that can be preset for gene expression timing is realized for the first time in the present invention. It can preset gene expression time in engineering bacteria. Its characteristics make it possible to preprogram the engineered strains to perform chronological expression of different metabolite pathway synthesis genes. , can be used to control the gene expression of engineering strains in biomedical fields such as the synthesis of natural products and antibiotics, and engineering bacteria for the treatment of environmental pollutants.

Description of drawings

Figure 1 is a schematic diagram of the principle that the initial conduction target in the fuse sequence activates its adjacent conduction targets and transforms them into the initial conduction target.

Figure 2 is a schematic diagram of the principle in which the excitation target point in the fuse sequence is activated by its adjacent conduction target point and thereby modifies the -35box of the tac promoter.

Figure 3 shows a schematic diagram of the relative positions of the priming target and promoter in the fuse sequence.

Figure 4 shows a schematic structural diagram of a fuse sequence including an initial conduction target, a conduction target (1) and an excitation target.

Figure 5 shows the pFuse1 plasmid map.

Figure 6 shows the pFuse2 plasmid map.

Figure 7 shows the pFuse3 plasmid map.

Figure 8 is the plasmid map of pACYC-ABE7.10(dCas9).

Figure 9 shows the colony status on the plates with different culture times.

Figure 10 shows the colony status on different plates under the same culture time (7 days).

Figure 11 shows the fluorescence intensity analysis of colonies on different plates under the same culture time (**** in the figure indicates a significant difference, P<0.0001).

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

The present invention designs a fuse sequence as a gene expression control switch element.

1. Design rules of fuse sequence

The overall organization of the design fuse sequence (which can also be called the overall sequence) is as follows:

5’-Tac promoter-adapter-[basic unit]×n-NBB-TGG-3’

Among them, the nucleotide sequence of the Tac promoter is shown in SEQ ID NO.1, and TGTCAA is the -35box of the Tac promoter;

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

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

Assume that the sum of the number of G and C in the three bases closest to the 5' end of the basic unit is x, and the sum of the number of G and C in all 17 bases of the basic unit is y, then x+y=10. Assume that the sum of the numbers of G and C in the 11 bases of the adapter segment is z, then x+z=8.

According to the different positions in the above-mentioned fuse sequence, there are three different sequences of ABE7.10 targets in the above-mentioned fuse sequence in the initial state:

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

(2) The conduction target (5’-NBB-TAABNNNNNNNNNNNNBB-TAA-3’), the middle part of the overall sequence is this sequence in the initial state. There is no active PAM at the 3' end of the conduction target, so it cannot be recognized and modified by ABE7.10 (dCas9) in its initial state. However, because the 3' end of the conduction target overlaps with the editing window of the initial conduction target, after the initial conduction target is edited by ABE7.10 (dCas9), the PAM sequence of the conduction target is converted into TGG, thus making the conduction target point is converted into a new initial conduction target.

(3) Excitation target (5'-TGTCAA-adapter-NBB-TAA-3'), which is the part of the overall sequence close to the Tac promoter. There is no active PAM at the 3' end of the excitation target, so it cannot be recognized and modified by ABE7.10 (dCas9) in its initial state. Similarly, since the 3' end of the excitation target overlaps with the editing window of the previous conduction target, when the previous conduction target is activated and converted into a new initial conduction target and modified by ABE7.10 (dCas9), the excitation target The PAM sequence of the dot will be converted into TGG, which can be recognized by ABE7.10 (dCas9), and the two outermost adenine bases of the -35 box of the Tac promoter will be modified, inactivating the Tac promoter.

The above-mentioned initial conduction target and conduction target share a sgRNA, whose sequence is: 5-NBBTAABNNNNNNNNNNNBB-gRNAscaffold-3’, which is called conduction sgRNA;

The above excitation target uses the sequence 5’-TGTCAA-Adapter-NBB-gRNAscaffold-3’ as sgRNA, which is called excitation sgRNA.

It can be seen that for the fuse sequence, in the initial state, only the initial conduction target can be modified by ABE7.10 (dCas9), and neither the conduction target nor the excitation target can be modified by ABE7.10 (dCas9). However, when ABE7.10 (dCas9) recognizes the target closest to the 3' end (the initial conduction target), it will modify the AA near the 5' end of the target to GG, because the 5' end of the initial conduction target is different from the conduction target. The 3' end of the target overlaps. The above modification activates the PAM of the conduction target, and the conduction target is converted into the initial conduction target, which can then be recognized by ABE7.10 (dCas9). Then the target is close to the 5' end. AA is modified to GG. Since the 5' end of this target overlaps with the 3' end of the next conduction target, the above modification causes the PAM of the next conduction target to be activated. By analogy, each modification of the target near the 5' end will activate the PAM of the next target, thereby activating each target in sequence from the 3' end to the 5' end of the entire sequence, until finally it is close to the adapter segment. The PAM of the triggering target of the promoter is activated. After the triggering target is recognized by ABE7.10 (dCas9), the two outermost adenine bases of the -35box of the Tac promoter are modified, inactivating the Tac promoter. , thereby achieving the purpose of regulating gene expression. In the above fuse sequence, the logic of sequential activation of each target is similar to that of a fuse, which is the origin of the name of the "fuse sequence".

Figure 1 is a schematic diagram of the principle that the initial conduction target closest to the 3' end in the fuse sequence activates its adjacent conduction target and converts it into an initial conduction target.

As shown in Figure 1, the AA in the editing window of the initial conduction target is in the area shared by the two targets. Therefore, the AA in the editing window of the initial conduction target is edited into GG by ABE7.10 (dCas9), which is equivalent to the conduction target. The inactivated PAM of the point is converted into an active PAM, and the conduction target is converted into the initial conduction target, so that it can be recognized by ABE7.10 (dCas9), and then the AA in the editing window of the conduction target is also recognized by ABE7. 10(dCas9) edited to GG.

In addition, although it is not shown in Figure 1, it is obvious that when the conduction target in Figure 1 is activated and converted into an initial conduction target, it and the next conduction target will also form a group as shown in Figure 1 The initial conduction target and the adjacent conduction target, so that the next conduction target will also be activated and converted into the initial conduction target. Each conduction target is stimulated in this sequence.

Figure 2 is a schematic diagram of the principle in which the excitation target point in the fuse sequence is activated by its adjacent conduction target point and thereby modifies the -35box of the tac promoter. Figure 3 shows a schematic diagram of the relative positions of the priming target and promoter in the fuse sequence.

As shown in Figure 2, the AA in the editing window of the conduction target is in the area shared by the two targets. Therefore, after the AA in the editing window of the conduction target is edited to GG by ABE7.10 (dCas9), the loss of the stimulation target Live PAM is converted into active PAM, and the excitation target can be recognized by ABE7.10 (dCas9). Then the two outermost adenine bases of the -35box of the Tac promoter are edited by ABE7.10 (dCas9), and Tac is started. Son is inactivated.

Figure 4 shows a schematic structural diagram of a fuse sequence including an initial conduction target, a conduction target (1) and an excitation target. In Figure 4, there is a common area between the initial conduction target point and the conduction target point, and there is a common area between the conduction target point and the excitation target point.

Of course, since the number n of basic units in the fuse sequence can be 1, 2, 3..., the fuse sequence can only include the initial conduction target point and the excitation target point (n=1) , it can also include initial conduction target, conduction target (1) and excitation target (n=2), or it can include initial conduction target, conduction target (multiple) and excitation target (n> 2). At this time, the number of conduction targets is adjustable, m=0,1,2,3...

It can be found that in the above fuse sequence, depending on the value of the number of basic units n, the length of the sequence is different, and the number of targets that can be edited by ABE7.10 in the initial state of the sequence is different. Then, by adjusting the number of basic units, the type and/or quantity of targets can be adjusted, and the relative time of gene expression regulation can be preset. For fuse sequences, the greater the number of targets, the longer the gene expression time. For a fuse sequence that contains an initial conduction target, a conduction target, and an excitation target at the same time, the greater the number of conduction targets, the longer the gene expression time. The fuse sequence can be used as a regulatory switch element for gene expression.

In order to more specifically illustrate the regulation of the temporal expression of the target gene by the fuse sequence, an example will be given below: select a basic unit and corresponding connecting segment that conforms to the above fuse sequence design rules to construct fuses of different lengths. fuse sequence, and the mCherry red fluorescent reporter gene was used as the target gene to characterize the excitation time differences of fuse sequences of different lengths.

2. Examples

(1) Overall sequence of fuses

According to the above rules, the basic units and connecting sections that meet the requirements are designed, as follows:

The nucleotide sequence of the basic unit is shown in SEQ ID NO.2; the nucleotide sequence of the adapter segment is shown in SEQ ID NO.3.

The overall sequence of fuses is:

5’-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTGACG-

[AGCTAACTGCAGTCACT]×n-NBB-TGG-3’

Among them, B represents a base among G, T, and C, N represents any base, n=1, 2, 3...

In the overall sequence of the above-mentioned fuse, the initial conduction target is located in the part closest to the 3' end of the overall sequence. The nucleotide sequence of the initial conduction target is shown in SEQ ID NO. 4; the conduction target is located in the middle of the overall sequence. The nucleotide sequence of the conduction 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 As shown in SEQ ID NO.6.

The initial conduction target and the conduction target share a conduction sgRNA. The nucleotide sequence of the conduction sgRNA is shown in SEQ IDNO.7, which can be written as: 5’-AGCTAACTGCAGTCACTAGC-gRNAscaffold-3’.

The sgRNA sequence used to excite the target is excitation sgRNA. The nucleotide sequence of the excitation sgRNA is shown in SEQ ID NO.8, which can also be written as: 5’-TGTCAATCATGCTGACGAGC-gRNAscaffold-3’.

(2) Design of three fuse sequences of different lengths

According to 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, and the Fuse3 sequence contains an An initial conduction target, a conduction target, and an excitation target.

In addition, for the convenience of comparison, a sequence containing only one excitation target was designed here. At the same time, in order to enable a single excitation target to be recognized by ABE7.10, the 3' TAA of the excitation target was modified to TGG. For convenience of explanation, this series is called Fuse1 sequence here.

Therefore, although the Fuse1 sequence is a specially designed comparison sequence and is different from the fuse sequences Fuse2 and Fuse3, in the following description, for the sake of convenience, the Fuse1 sequence, Fuse2 sequence and Fuse3 sequence are collectively referred to as Three different length fuse sequences.

The corresponding sequences are as follows:

Fuse1 sequence: 5’-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTG ACG-NBB-TGG-3’;

Fuse2 sequence: 5’-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTG ACG-AGCTAACTGCAGTCACT-NBB-TGG-3’;

Fuse3 sequence: 5’-CATTATACGAGCCGATGATTAAT-TGTCAA-TCATGCTG ACG-AGCTAACTGCAGTCACT-AGCTAACTGCAGTCACT-NBB-TGG-3’;

B represents one of G, T, and C bases, and N represents any base.

(3) Trigger sequences are used for temporal regulation of gene expression

3.1 Instructions for raw materials

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

3.2 Sequence synthesis

Beijing Qingke Biotechnology Co., Ltd. was entrusted to synthesize the following fragments: mCherry protein fragment, conductive sgRNA fragment, excitation sgRNA fragment, Fuse1 fragment, Fuse2 fragment, and Fuse3 fragment.

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

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

The nucleotide sequence of the priming sgRNA fragment is shown in SEQ ID NO. 11. This fragment contains the priming sgRNA as well as fragments with low homology to other components to facilitate recombination.

The nucleotide sequence of the Fuse1 fragment is shown in SEQ ID NO. 12. This fragment contains the reserved one-step cloning homology region (the first 21 positions), the antisense strand sequence of the Fuse1 sequence (antiparallel to the Fuse1 sequence) and the Other components are fragments with lower homology to facilitate recombination.

The nucleotide sequence of the Fuse2 fragment is shown in SEQ ID NO. 13. This fragment contains the reserved one-step cloning homology region (the first 21 positions), the antisense strand sequence of the Fuse2 sequence (antiparallel to the Fuse1 sequence) and the Other components are fragments with lower homology to facilitate recombination.

The nucleotide sequence of the Fuse3 fragment is shown in SEQ ID NO. 14. This fragment contains the reserved one-step cloning homology region (the first 21 positions), the antisense strand sequence of the Fuse3 sequence (antiparallel to the Fuse1 sequence) and the Other components are fragments with lower homology 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 Schein-Dalgarno sequence). The tac promoter and ribosome binding site ensure that the subsequent mCherry protein sequence can be transcribed and translated normally.

3.3 Plasmid construction

(1) pFuse1 plasmid construction:

Use the first primer CD1 and the second primer CD2 to amplify the conducting sgRNA fragment, use the third primer Fuse-1 and the fourth primer Fuse-2 to amplify the Fuse1 fragment, use the fifth primer mCherry1 and the sixth primer mCherry2 to amplify the mCherry fragment, Plasmid pCDFDuet-1 was amplified using the seventh primer pCDFDuet1-1 and the eighth primer pCDFDuet1-2.

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, and the nucleotide sequence of the third primer Fuse-1 is shown in SEQ ID NO.15. ID NO.17 is shown. 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 core sequence of the sixth primer mCherry2 is shown in SEQ ID NO.18. The nucleotide sequence 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.

Amplified using Transgen’s FastPfu Fly DNA Polymerase. The amplification system includes: 1 μl of FastPfu Fly DNA Polymerase, 10 μl of Fly Buffer, 6 μl of 2.5mM dNTPs, 30 μl of deionized water, 1 μl of each primer, and 1 μl of template. The PCR program is as follows: (1) 95°C, 3 minutes; (2) 95°C, 30 seconds; (3) 58°C, 30 seconds; (4) 72°C, 2 minutes; repeat steps (2) to (4) 35 cycles; (5) 72°C, 5 minutes.

Use the amplified PCR product using Vazyme's The Ultra One Step CloningKit kit performs one-step cloning. After mixing the above four amplified fragments (conduction sgRNA fragment, Fuse1 fragment, mCherry fragment and plasmid pCDFDuet-1) according to the molecular weight of 1:1:1:1, add an equal amount of 2×ClonExpress Mix, connect in a 50°C water bath for 1 hour, cool on ice for 5 minutes, take 10 μl of Transgen-added E. coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, and cool in a 42°C water bath Heat shock in the pot for 75 seconds, add 500 microliters of LB liquid culture medium, incubate for two hours on a shaker at 37°C at 220rpm, spread onto a LB medium plate containing streptomycin, invert the culture at 37°C for 24 hours, and then pick and sequence the bacteria. After picking the correct colony, the pFuse1 plasmid construction is completed. The pFuse1 plasmid map is shown in Figure 5.

(2) pFuse2 plasmid construction:

The same as the pFuse1 plasmid construction, use the first primer CD1 and the second primer CD2 to amplify the conducting sgRNA fragment, use the third primer Fuse-1 and the fourth primer Fuse-2 to amplify the Fuse2 fragment, use the fifth primer mCherry1 and the sixth primer mCherry2 amplifies the mCherry fragment, and uses the seventh primer pCDFDuet1-1 and the eighth primer pCDFDuet1-2 to amplify plasmid pCDFDuet-1.

Amplified using Transgen’s FastPfu Fly DNA Polymerase. The amplification system includes: 1 μl of FastPfu Fly DNA Polymerase, 10 μl of Fly Buffer, 6 μl of 2.5mM dNTPs, 30 μl of deionized water, 1 μl of each primer, and 1 μl of template. The PCR program is as follows: (1) 95°C, 3 minutes; (2) 95°C, 30 seconds; (3) 58°C, 30 seconds; (4) 72°C, 2 minutes; repeat steps (2) to (4) 35 cycles; (5) 72°C, 5 minutes.

Use the amplified PCR product using Vazyme's The Ultra One Step CloningKit kit performs one-step cloning. After mixing the above four fragments (conducting sgRNA fragment, Fuse2 fragment, mCherry fragment and plasmid pCDFDuet-1) according to the molecular weight of 1:1:1:1, add an equal amount of 2× ClonExpress Mix, after connecting in a 50°C water bath for 1 hour, cool on ice for 5 minutes, take 10 μl of E. coli BL21 (DE3) competent cells added to Transgen, incubate on ice for 30 minutes, and place in a 42°C water bath. Heat shock for 75 seconds, add 500 microliters of LB liquid culture medium, incubate at 37°C shaker at 220rpm for two hours, spread on the LB medium plate containing streptomycin, invert the culture at 37°C for 24 hours, pick the bacteria for sequencing, and pick out After the correct colony is created, the pFuse2 plasmid construction is completed. The pFuse2 plasmid map is shown in Figure 6.

(3) pFuse3 plasmid construction:

The same as the pFuse1 plasmid construction, use the first primer CD1 and the second primer CD2 to amplify the conducting sgRNA fragment, use the third primer Fuse-1 and the fourth primer Fuse-2 to amplify the Fuse3 fragment, use the fifth primer mCherry1 and the sixth primer mCherry2 amplifies the mCherry fragment, and uses the seventh primer pCDFDuet1-1 and the eighth primer pCDFDuet1-2 to amplify plasmid pCDFDuet-1.

Amplified using Transgen’s FastPfu Fly DNA Polymerase. The amplification system includes: 1 μl of FastPfu Fly DNA Polymerase, 10 μl of Fly Buffer, 6 μl of 2.5mM dNTPs, 30 μl of deionized water, 1 μl of each primer, and 1 μl of template. The PCR program is as follows: (1) 95°C, 3 minutes; (2) 95°C, 30 seconds; (3) 58°C, 30 seconds; (4) 72°C, 2 minutes; repeat steps (2) to (4) 35 cycles; (5) 72°C, 5 minutes.

Use the amplified PCR product using Vazyme's The Ultra One Step CloningKit kit performs one-step cloning. After mixing the above four amplified fragments (conducting sgRNA fragment, Fuse3 fragment, mCherry fragment and plasmid pCDFDuet-1) according to the molecular weight of 1:1:1:1, add an equal amount of 2×ClonExpress Mix, connect in a 50°C water bath for 1 hour, cool on ice for 5 minutes, take 10 μl of Transgen-added E. coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, and cool in a 42°C water bath Heat shock in the pot for 75 seconds, add 500 microliters of LB liquid culture medium, incubate on a 37°C shaker at 220rpm for two hours, spread on the LB medium plate containing streptomycin, invert the culture at 37°C for 24 hours, then pick the bacteria for sequencing. After picking the correct colony, the pFuse3 plasmid construction is completed. The pFuse3 plasmid map is shown in Figure 7.

(4) pJF plasmid construction:

The ninth primer JF1 and the tenth primer JF2 were used to amplify the excitation sgRNA fragment, and the eleventh primer pACYCDuet1-1 and the twelfth primer pACYCDuet1-2 were used to amplify the plasmid pACYCDuet-1.

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, and the nucleotide sequence of the eleventh primer pACYCDuet1-1 is shown in SEQ ID NO. 25 is shown, and the nucleotide sequence of the twelfth primer pACYCDuet1-2 is shown in SEQ ID NO. 26.

Amplified using Transgen’s FastPfu Fly DNA Polymerase. The amplification system includes: 1 μl of FastPfu Fly DNA Polymerase, 10 μl of Fly Buffer, 6 μl of 2.5mM dNTPs, 30 μl of deionized water, 1 μl of each primer, and 1 μl of template. The PCR program is as follows: (1) 95°C, 3 minutes; (2) 95°C, 30 seconds; (3) 58°C, 30 seconds; (4) 72°C, 2 minutes; repeat steps (2) to (4) 35 cycles; (5) 72°C, 5 minutes.

Use the amplified PCR product using Vazyme's The Ultra One Step CloningKit kit performs one-step cloning. Mix the above two amplified fragments (stimulating sgRNA fragment and plasmid pACYCDuet-1) at a molecular weight of 1:1, add an equal amount of 2×ClonExpress Mix, and incubate in a 50°C water bath. After ligation for 1 hour, cool on ice for 5 minutes, absorb 10 μl of E. coli BL21 (DE3) competent cells added with Transgen, incubate on ice for 30 minutes, heat shock in a 42°C water bath for 75 seconds, add 500 μl Literally increase the LB liquid medium, culture it on a shaker at 220 rpm at 37°C for two hours, spread it onto the LB medium plate containing streptomycin, invert it at 37°C for 24 hours, then select the bacteria for sequencing. After picking the correct bacteria, the pJF plasmid is constructed. .

(5) pACYC-ABE7.10 plasmid construction:

The thirteenth primer ABE7.10-1 and the fourteenth primer ABE7.10-2 were used to amplify the ABE7.10 plasmid, and the fifteenth primer pJF-1 and the sixteenth primer pJF-2 were used to amplify the plasmid pJF.

The nucleotide sequence of the thirteenth primer ABE7.10-1 is shown in SEQ ID NO.27, the nucleotide sequence of the fourteenth primer ABE7.10-2 is shown in SEQ ID NO.28, and the fifteenth primer The nucleotide sequence of pJF-1 is shown in SEQ ID NO. 29, and the nucleotide sequence of the sixteenth primer pJF-2 is shown in SEQ ID NO. 30.

Amplified using Transgen's FastPfu Fly DNA Polymerase. The amplification system includes: 1 μl of FastPfu Fly DNA Polymerase, 10 μl of Fly Buffer, 6 μl of 2.5mM dNTPs, 30 μl of deionized water, 1 μl of each primer, and 1 μl of template. The PCR program is as follows: (1) 95°C, 3 minutes; (2) 95°C, 30 seconds; (3) 58°C, 30 seconds; (4) 72°C, 2 minutes; repeat steps (2) to (4) 35 cycles; (5) 72°C, 5 minutes.

Use the amplified PCR product using Vazyme's The Ultra One Step CloningKit kit performs one-step cloning. Mix the above two amplified fragments (ABE7.10 plasmid and pJF plasmid) at a molecular weight of 1:1, add an equal amount of 2×ClonExpress Mix, and incubate in a 50°C water bath. After 1 hour of connection, cool on ice for 5 minutes, take 10 μl of E. coli BL21 (DE3) competent cells added to Transgen, incubate on ice for 30 minutes, heat shock in a 42°C water bath for 75 seconds, add 500 μl LB liquid culture medium, culture at 37°C shaker at 220rpm for two hours, spread onto LB medium plate containing streptomycin, invert culture at 37°C for 24 hours, then pick the bacteria for sequencing. After picking the correct bacteria, pACYC-ABE7. 10. Plasmid construction is completed.

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

The seventeenth primer H840A-1 and the eighteenth primer H840A-2 were used to amplify the pACYC-ABE7.10 plasmid. The nucleotide sequence of the seventeenth primer H840A-1 is shown in SEQ ID NO.31. The eighteenth primer The nucleotide sequence of primer H840A-2 is shown in SEQ ID NO. 32.

The amplification system includes: 1 μl of FastPfu Fly DNA Polymerase, 10 μl of Fly Buffer, 6 μl of 2.5mMdNTPs, 30 μl of deionized water, 1 μl of each primer, and 1 μl of template. The PCR program is as follows: (1) 95°C, 3 minutes; (2) 95°C, 30 seconds; (3) 58°C, 30 seconds; (4) 72°C, 2 minutes; repeat steps (2) to (4) 25 cycles; (5) 72°C, 5 minutes.

Add 1 microliter of DMT enzyme from Transgen's Fast Mutagenesis System to the product, put the mixture into a 37°C water bath and incubate for 1 hour, then take 10 microliters of Transgen's E. coli BL21 (DE3) competent cells and incubate on ice for 30 minutes, heat shock in a 42°C water bath for 75 seconds, add 500 μl of LB liquid culture medium, incubate on a 37°C shaker at 220 rpm for two hours, spread onto an LB medium plate containing streptomycin, and incubate upside down at 37°C for 24 Hours later, the bacteria were picked and sequenced. After the correct bacteria were picked, the pACYC-ABE7.10 (dCas9) plasmid was constructed. This process can also be referred to as the H840A point mutation of pACYC-ABE7.10(dCas9). The plasmid map of pACYC-ABE7.10(dCas9) is shown in Figure 8.

3.4 Transformation of E. coli with plasmid DNA

Add 200 ng of pFuse1 plasmid and 250 ng of pACYC-ABE7.10 (dCas9) plasmid to Transgen's Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, heat shock at 42°C in a water bath for 75 seconds, and add 500 microliters of LB liquid culture medium was cultured for two hours on a shaker at 220 rpm at 37°C, and spread on an LB medium plate containing chloramphenicol and streptomycin for growth. After incubation at 37°C for a period of time, observe the plate 1 .

Add 200 ng of pFuse2 plasmid and 250 ng of pACYC-ABE7.10 (dCas9) plasmid to Transgen's Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, heat shock at 42°C in a water bath for 75 seconds, and add 500 microliters of LB liquid culture medium was cultured for two hours on a shaker at 220 rpm at 37°C, and spread on an LB medium plate containing chloramphenicol and streptomycin for growth. After incubation at 37°C for a period of time, observe the plate 2 .

Add 200 ng of pFuse3 plasmid and 250 ng of pACYC-ABE7.10 (dCas9) plasmid to Transgen's Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, heat shock at 42°C in a water bath for 75 seconds, and add 500 microliters of LB liquid culture medium was cultured for two hours on a shaker at 220 rpm at 37°C, and spread on an LB medium plate containing chloramphenicol and streptomycin for growth. After incubation at 37°C for a period of time, observe the plate 3 .

Control group: Add 200 ng of pCDFDuet-1 plasmid and 250 ng of pACYC-ABE7.10 (dCas9) plasmid to Transgen's Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, and heat in a water bath at 42°C. Stimulate for 75 seconds, add 500 microliters of LB liquid culture medium, incubate for two hours on a 37°C shaker at 220rpm, and spread on an LB medium plate containing chloramphenicol and streptomycin for growth, and incubate at 37°C for a period of time. Finally, observe plate 4.

3.5 Results

When the culture time is 2 days, 3 days, 4 days, 5 days, and 7 days, observe plate 3 and record the growth of the colonies, as shown in Figure 9. Figure 9 records the development process of the chimeric colony expressing red fluorescent protein on plate 3. The dark area of the colony is the red color of mCherry expression, and the light area of the colony is the milky white color of the control colony (E. coli).

As can be seen from Figure 9, when the culture time is short, the colony as a whole is red with mCherry expression; as the culture time increases, the newly grown parts around the colony appear milky white without mCherry expression, and, within a period of time, After a while, the milky white bacteria completely surrounded the outer ring of the colony. This is because the genes controlled by the regulatory element switch will be inactivated after a period of time, that is, the mCherry gene will stop expression after a period of time. Therefore, there will be two phenotypes in the colonies growing on the plate, one expresses mCherry and appears red; the other does not express mCherry and appears milky white. Because they grow on a plate, the bacterial cells on the periphery of the colony are relatively new and tend to show a white phenotype that does not express mCherry with delayed gene expression time; while the bacteria in the center of the colony grow earlier and tend to show earlier gene expression time. Expresses the red phenotype of mCherry.

Under the same culture time (7 days), observe plate 1, plate 2 and plate 3, as shown in Figure 10. It can be found that the colony performance of the three is different: the overall color of the colonies on plate 3 is more reddish, the overall color of the colonies on plate 1 is whiter, and the overall color of the colonies on plate 2 is somewhere in between. This shows that under the same culture time (7 days), the colony carrying pFuse3 expresses mCherry for the longest time, and there will be more areas expressing mCherry on plate 3; the colony carrying pFuse1 expresses mCherry for the shortest time, and on plate 3 the mCherry expression time is the shortest. The area expressing mCherry on plate 1 will be less; the expression of mCherry in the colony carrying pFuse2 is somewhere in between, and the area expressing mCherry on plate 2 is also somewhere in between. The reason is that pFuse3 has the most targets, pFuse1 has the least targets, and pFuse2 has targets in between. Different numbers of targets result in different conduction activation times, which lead to different times for reaching and activating the excitation target and then modifying the two outermost adenine bases of the -35box of the Tac promoter to inactivate the Tac promoter. Therefore, The length of time mCherry is expressed varies. This shows that regulatory elements containing three different lengths of fuse sequences (pFuse1, pFuse2, and pFuse3) have different numbers of targets and different lengths of mCherry expression. That is, the temporal regulation of mCherry gene expression can be achieved by adjusting the number of targets in the fuse sequence. For example, when the number of conduction targets in the fuse sequence increases, the time for expressing mCherry in colonies carrying the fuse sequence becomes longer.

In order to characterize the difference in total mCherry expression among the above-mentioned colonies equipped with fuses of different lengths, fluorescent characterization was performed on the above three types of plates and the control plate. Wash all the colonies on each plate that have been grown for 7 days with 1×PBS buffer, and dilute to OD600 = 1.0. In a standard 96-well plate with a transparent bottom, use the colonies on plate 4 in the control group that do not express mCherry. The colony of BL21(DE3) was used as a control, and its fluorescence intensity was measured using the SpectraMax M5 microplate reader from Molecular Divices. The excitation light wavelength is 587nm, the emission light wavelength is 610nm, the sample volume is 100 microliters, and the number of repetitions is 3 times, and the fluorescence intensity data shown in Figure 11 is obtained.

It can be seen from Figure 11 that the fluorescence intensity of colonies carrying pFuse3 is higher than that of colonies carrying pFuse2 and pFuse1, indicating that the longer the fuse sequence, the more the reporter gene (mCherry red fluorescent gene) is expressed, and the reporter gene is inactivated. The slower the speed. Because in the inactive state, the tac promoter of the gene regulatory element is not modified, mCherry fluorescent protein is expressed normally and displays red fluorescence in E. coli; in the activated state, the tac promoter of the gene regulatory element is modified, and mCherry expression is lost. alive, the red fluorescence disappears. This shows that at the same concentration levels of ABE7.10(dCas9), excitation sgRNA and conduction sgRNA, the shorter the length of the fuse sequence, the shorter the time required for mCherry to stop expression. Therefore, gene expression timing can be controlled by adjusting the length of the fuse sequence. This proves that the purpose of regulating the timing of gene expression can be achieved by pre-programming the length of the fuse sequence of the regulatory element.

Based on the same principle, any fuse sequence that complies with the design rules in the embodiments of the present invention can achieve the above function of sequential regulation of gene expression. The basic unit and adapter segment are by no means limited to one of the examples. Under the design rules of the present invention, they can be adjusted as needed so that dCas9 binds to the target matched by the sgRNA through the sgRNA. Moreover, the number of targets (i.e., the length of the fuse sequence) can also be adjusted according to actual requirements.

At the same time, the tac promoter used in the present invention is a universal promoter in E. coli and can efficiently activate 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.

Therefore, the applications of the above-mentioned gene expression control elements or systems include, but are not limited to: pre-programming of temporal sequential expression of different metabolite pathway synthesis genes of engineered strains; engineered strains for the synthesis of natural products and antibiotics Gene expression control; gene expression control for engineering bacteria that synthesize environmental pollutants.

sequence list

<110> Zhejiang University

<120> Gene temporal expression regulation system and 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> 23

<212> DNA

<213> Artificial Sequence

<400> 4

agctaactgc agtcactagc tgg 23

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 5

agctaactgc agtcactagc taa 23

<210> 6

<211> 23

<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> Red fluorescent protein (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

gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360

ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660

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

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

cactagctgggaattcggatccgcgacccattacc 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> 21

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 21

tagctgtcaa cctaatgcag gagtcgcata aggggag 36

<210> 22

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 22

gtacaagtaa caaagcccga aaggaagctg agttg 35

<210> 23

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 23

ctgcattagg ttgacagcta gctcagtcct aggtataata ctagt 45

<210> 24

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 24

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 (10)

1. A gene expression control switching element characterized in that the overall sequence of the gene expression control switching element is as follows:

5'-Tac promoter-adapter segment- [ basic Unit ]. Times.n-NBB-TGG-3'

Wherein,

the sequence of the basic units is as follows: 5 '-NBBTASNNNNNNNNNNNNN-3';

the sequence of the linking segment (linker) is as follows: 5'-NNNNNNNNNNN-3';

the nucleotide sequence of the Tac promoter is shown as SEQ ID NO.1, wherein TGTCAA is-35 box of the Tac promoter;

b represents one base in G, T, C, N represents any base, N is the number of basic units, n=1, 2,3 … …;

assuming that the sum of the numbers of G and C in the 3 bases closest to the 5' end of the basic unit is x, and the sum of the numbers of G and C in all 17 bases of the basic unit is y, then x+y=10; let the sum of the numbers of G and C in all 11 bases of the adapter be z, then x+z=8.

2. The gene expression regulatory switching element of claim 1, wherein the gene expression regulatory switching element has 1 initial conduction target, m conduction targets, and 1 excitation target in an initial state, m = 0,1,2,3 … …; wherein,

the initial conduction target is positioned at the part of the overall sequence closest to the 3' end, and the sequence of the initial conduction target is as follows: 5'-NBB-TAABNNNNNNNNNNNBB-TGG-3';

the conduction target is positioned in the middle section of the total sequence, and the sequence of the conduction target is as follows: 5 '-NBB-TAABNNNNNNNNNBB-TAA-3';

the excitation target is positioned on a part of the total sequence close to the Tac promoter, and the sequence of the excitation target is as follows: 5 '-TGTCAA-adapter segment-NBB-TAA-3';

the initial and the conduction targets share one sgRNA, called the conduction sgRNA, and the sequence of the conduction sgRNA is: 5-NBBTASNNNNNNNNNNNBB-gRNAscafold-3';

the sgRNA used for the priming target is called the priming sgRNA, and the sequence of the priming sgRNA is 5 '-TGTCAA-adapter segment-NBB-gRNAscafold-3'.

3. The gene expression control switching element according to claim 1, wherein 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 adapter is shown in SEQ ID NO. 3; the general sequence of the gene expression control switching element is as follows:

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

Wherein B represents one base in G, T, C, N represents any base, and n=1, 2,3 … ….

4. The gene expression control switching element according to claim 3,

the gene expression regulation and control switch element has 1 initial conduction target point, m conduction target points and 1 excitation target point in an initial state, wherein m=0, 1,2,3 and … …; wherein,

the initial conduction target is positioned at the part of the overall sequence closest to the 3' end, and the nucleotide sequence of the initial conduction target is shown as SEQ ID NO. 4; the conduction target is positioned in the middle section of the total sequence, the nucleotide sequence of the conduction target is shown as SEQ ID NO.5, and m=0, 1,2,3 and … …; the excitation target is positioned at a part of the total sequence close to the Tac promoter, and the nucleotide sequence of the excitation target is shown as SEQ ID NO. 6;

the initial conduction target and the conduction target share one sgRNA, which is called as conduction sgRNA, and the nucleotide sequence of the conduction sgRNA is shown as SEQ ID NO. 7; the sgRNA sequence used for exciting the target is excited sgRNA, and the nucleotide sequence of the excited sgRNA is shown as SEQ ID NO. 8.

5. A gene expression control system comprising: comprising the following steps: pFUuse plasmid and pACYC-ABE7.10-dCAS9 plasmid, wherein,

The pFuse plasmid is obtained by the following method: cloning a preset length of the gene expression control switching element according to any one of claims 1 to 4 into a pcdfdurt-1 plasmid to obtain a detonating cord plasmid; cloning the target gene and the conduction sgRNA to the detonating cord plasmid to obtain a pFUuse plasmid;

the pACYC-ABE7.10-dCAS9 plasmid is obtained by the following method: mutating the 840 th residue histidine point of ABE7.10 into alanine to obtain ABE7.10-dCAS9; and cloning the ABE7.10-dCAs9 and the stimulated sgRNA into pACYCDuet-1 to obtain pACYC-ABE7.10-dCAs9 plasmid.

6. The gene expression control system of claim 5, wherein the gene of interest comprises a functional gene or a regulatory factor.

7. A method of gene expression regulation comprising:

(1) Designing and synthesizing a predetermined length of the gene expression control switching element according to any one of claims 1 to 4, cloning the gene expression control switching element into a pcdfdurt-1 plasmid, thereby constructing a fuse plasmid, cloning a target gene and the conductance sgRNA into the fuse plasmid, and obtaining a pFuse plasmid;

(2) Mutating the 840 th residue histidine point of ABE7.10 into alanine to obtain ABE7.10-dCAS9; and cloning the ABE7.10-dCAs9 and the stimulated sgRNA into pACYCDuet-1 to obtain pACYC-ABE7.10-dCAs9 plasmid.

(3) The pFuse plasmid and the pACYC-ABE7.10-dCas9 plasmid are simultaneously transformed into recipient cells to regulate expression of the gene of interest.

8. The method of claim 7, wherein in step (3), the pFuse plasmid and the pACYC-ABE7.10-dCas9 plasmid are simultaneously transformed into competent cells of escherichia coli to regulate expression of the target gene.

9. The method of gene expression regulation of claim 7, wherein the gene of interest comprises a functional gene or a regulatory factor.

10. The use of a gene expression control system according to claim 5, including but not limited to: the method comprises the steps of pre-programming sequential expression of different metabolite pathway synthesis genes for engineering strains; control of gene expression in engineering strains for the synthesis of natural products and antibiotics; the method is used for controlling the gene expression of the engineering bacteria for synthesizing the environmental pollutant treatment.