WO2022126460A1

WO2022126460A1 - Gene element and use method thereof

Info

Publication number: WO2022126460A1
Application number: PCT/CN2020/137025
Authority: WO
Inventors: 傅雄飞; 储攀; 朱静雯; 程思; 何彩云
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-06-23

Abstract

A gene element and a use method thereof. The gene element is used for regulating the expression of a gene, and comprises: a transcription factor sequence provided upstream of the gene for coupling the expressions of the transcription factor and the gene; and a promoter sequence provided upstream of the transcription factor sequence for initiating the expression of the sequence downstream of the promoter. The gene element has the following advantages: the gene element does not rely on any inducing conditions, including chemical molecules and physical conditions; the whole process requires no human supervision; and the gene circuit is simple, and has low costs and high efficiency when reconstructed and deployed to different chassis cells or applied to different fermentation products.

Description

A genetic element and method of using the same

technical field

The present invention relates to a genetic element and a method of using the genetic element.

Background technique

At present, human beings have realized the production of large amounts of energy, chemicals and drugs through technologies such as gene editing that enable cell factories to convert autogenous endogenous energy and metabolites into pre-designed metabolites. However, in the process of producing such exogenous products, it will inevitably compete with cells for resources, and even become toxic to the cells themselves, resulting in the destruction of the normal cell structure or the disorder of the metabolic network. Failure, cell death, yield reduction, waste of energy and carbon sources, etc.

By dynamically regulating the gene expression of cells in the metabolic process, the utilization rate of substrates and the yield of products can be effectively improved, and the metabolic pathways of cells can be dynamically regulated. Metabolic imbalance. For example, exogenous gene circuits can be regulated at different stages of fermentation by externally responsive promoters such as inducers (IPTG, L-arabinose, anhydrotetracycline) responsive promoters, light, pH, dissolved oxygen, temperature responsive promoters The expression of the exogenous circuit can be coupled with the physiological state of the cell through the transcription factor that responds to the internal resources of the cell, so that the expression of the exogenous circuit is dependent on the physiological state of the cell, and the expression switch of the exogenous circuit is automatic and programmable.

The current class of dynamic regulation depends on the external conditions of the cell growth environment, conditional transcription mediated by inducers, temperature, light, dissolved oxygen, and pH-responsive promoters. Such pathways often require a large number of inducer molecules, increasing the It reduces the cost of the fermentation process; in addition, it is necessary to add the monitoring of the bacterial growth state during the fermentation process, which increases the cost of equipment and labor.

Another type of dynamic regulation strategy is to couple the cell's own metabolic processes, such as transcription factors that respond to intracellular precursor metabolites. Such transcription factors specifically respond to the concentration of intracellular precursors. When the concentration of intracellular precursors increases. When a certain threshold is reached, the expression of exogenous circuits is turned on. Such transcription factors are often endogenous proteins of cells, so they not only control the transcription of exogenous gene circuits, but also inevitably start the transcription of other metabolic pathways in the cell. On the one hand, it causes competition for intracellular resources, and on the other hand, it will lead to unpredictable metabolic changes; at the same time, the coupling of exogenous gene circuits and endogenous metabolic pathways involves a lot of trial-and-error process of editing the cell genome, which is difficult and takes a long time to develop.

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to provide a gene element capable of self-activation and dynamic regulation of gene expression using the global resource regulation mechanism in the cell.

The present disclosure provides a gene element for regulating the expression of a gene, having: a transcription factor sequence, the expression of which is configured to be coupled with the expression of the gene; a promoter sequence, which is configured upstream of the transcription factor sequence, for initiating Sequences downstream of the promoter are expressed. The gene element is suitable for expressing foreign proteins or enzymes with specific activities for the production of non-protein products.

Wherein, the transcription factor sequence is configured to express a transcription factor with constitutive transcriptional activity, such that the transcription factor is transcriptionally active in the absence of an inducer. The gene element is suitable for expressing foreign proteins or enzymes with specific activities for the production of non-protein products.

Among them, the transcription factor sequence is configured to express the AraC/xylS family protein whose N-terminus has a conformation after binding with L-arabinose.

Wherein, the transcription factor includes AraC protein, rhaS protein or rhaR protein with constitutive transcriptional activity.

Wherein, the transcription factor is AraC-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.1.

Wherein, the transcription factor is AraC-2 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.2.

Wherein, the transcription factor is AraC-3 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.3.

Wherein, the transcription factor is AraC-4 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.4.

Wherein, the transcription factor is AraC-5 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.5.

Wherein, the transcription factor is rhaS-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.6.

Wherein, the transcription factor is rhaR-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.7.

Wherein, the promoter sequence is configured as a wild-type Pbad promoter sequence or a wild-type Prhas promoter sequence.

Wherein, the promoter sequence is configured as the Pbad-1 promoter sequence after deleting the Crp binding site upstream of the wild-type Pbad promoter sequence, and the nucleotide sequence of the Pbad-1 promoter sequence is SEQ ID No. 8.

Wherein, the promoter sequence is configured as the Prhas-1 promoter after deleting the Crp binding site upstream of the wild-type Prhas promoter sequence, and the nucleotide sequence of the Prhas-1 promoter sequence is SEQ ID No. 9.

Wherein, the gene element includes Pbad promoter and AraC-1 protein, and the nucleotide sequence of the gene element is SEQ ID No.10.

Among them, genetic elements are configured to regulate the expression of genes or gene clusters.

Among them, a terminator is also included, and the terminator is arranged in the upstream or downstream sequence of the gene element to suppress transcriptional interference.

Among them, a fluorescent reporter gene is also included, and the fluorescent reporter gene is configured in the downstream sequence of the gene element to characterize the expression level of the gene or gene cluster.

The present disclosure provides a method for using a gene element, comprising: culturing cells equipped with the gene element in a medium; when the concentration OD600 of the cells exceeds 0.2, collecting the cells and inoculating the diluted cells into a new medium continue to cultivate.

The present disclosure provides a method for using a gene element, comprising: culturing cells equipped with the gene element in a medium; when the concentration OD600 of the cells exceeds 0.2, collecting the cells and inoculating the diluted cells into a new medium Culture in medium; when the cell concentration OD600 is close to 0.2, the new medium is supplemented with the precursors of exogenous products of cells and the culture is continued.

Wherein, the gene element is arranged upstream of the sequence of the gene or gene cluster to be regulated by a traceless assembly method.

Among them, the gene element is used by arranging the gene element in the genome or plasmid of the cell by the gene assembly method.

Among them, the sequence of the gene element, the sequence consisting of the same promoter sequence as the gene element and the sequence of the gene or gene cluster to be regulated in series are arranged in the same cell for use.

The cells are configured as one of Escherichia coli cells, Bacillus subtilis, lactic acid bacteria, Streptomyces, yeast, Corynebacterium, and animal cells.

Wherein, the medium is configured as one or more of LB medium, SOB medium, YT medium, TB medium, and RDM medium.

As can be seen from the above technical solutions, the present disclosure has the following advantages:

1. Compared with traditional inducible regulation, the present invention does not depend on any inducing conditions, including chemical molecules and physical conditions;

2. The whole process does not need manual supervision, saving costs;

3. It is suitable for all kinds of culture medium and has no dependence on the components of the medium;

4. Compared with the control circuit of the coupling of metabolic precursor molecules, the present invention is not directly coupled with the metabolism of cells, and has good orthogonality, so it can be directly used in the fermentation of different exogenous products through simple transformation, and the repeatability, high success rate;

5. The gene circuit is very simple, low cost and high efficiency when it is transformed and deployed to different chassis cells or applied to different fermentation products.

Description of drawings

1 is a schematic diagram of the structure of a gene element according to an embodiment of the disclosure;

Figure 2 is a graph of the relationship between cell growth rate and gene expression level according to an embodiment of the present disclosure;

FIG. 3 is a graph of the relationship between cell growth rate and gene expression level in the system of an embodiment of the present disclosure;

Figure 4 is the effect of different media on gene expression levels in one embodiment of the present disclosure.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present application more clearly understood, the present application will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

The present disclosure provides a gene element for regulating the expression of a gene, comprising: a transcription factor sequence configured upstream of the gene for coupling the transcription factor with the expression of the gene; a promoter sequence configured in the Upstream of transcription factor sequences, used to initiate expression of sequences downstream of a promoter. After the promoter sequence and the transcription factor sequence are connected in series, the gene element is arranged upstream of the gene, and the transcription factor sequence is located between the promoter sequence and the downstream gene. The gene element is suitable for expressing foreign proteins or enzymes with specific activities for the production of non-protein products.

It is understood in the present disclosure that cells have a global resource regulation mechanism in the growth process, and the global resource regulation mechanism will regulate the growth rate and regulate protein components according to the external nutritional status. For the production of ribosomes; and when external resources are scarce, the cell allocates a large amount of resources to the production of metabolism-related proteins. Therefore, the concentration of some constitutively expressed and cAMP-responsive protein products will increase significantly in the case of slow cell growth. The background expression level of the self-activation pathway can be changed within a certain range, so that when the cell growth rate is fast, the self-activation circuit is in a closed state, and it is automatically activated when the growth rate is lower than a certain threshold. The self-activation network motif, whose basic structure is a transcription factor capable of self-activation and a specific promoter that controls the transcription of the transcription factor, has the following characteristics: when the transcription factor is activated, it can rapidly promote the transcription of the transcription factor itself. And translation, to achieve rapid expression of the function of the downstream gene of the promoter within minutes. At the same time, the self-activating motif is bistable. Under a certain background expression level of transcription factors, the individual cells with the self-activating motif will appear in two states, namely the activated state and the inactive state. These states are randomly determined, resulting in two cell phenotypes in a population of cells carrying the same gene. When the background transcription level of the transcription factor is too low, the self-activating motif can only appear in the inactive state, and when the background transcription level is too high, the self-activating motif can only appear in the activated state. Due to the existing dynamic regulation or external conditions that depend on the cell growth environment, or coupled with the cell's self-generated metabolic process, each has its own defects.

The present disclosure utilizes the above information to design a gene element, wherein the transcription factor sequence is arranged between the promoter sequence of the transcription factor sequence and the downstream gene, and the expression of the transcription factor is coupled with the expression of the gene. During the transcription process, the promoter sequence is recognized by RNA polymerase and begins to transcribe the downstream transcription factor sequence and gene sequence. The expression level of the transcription factor will affect its own activation state and then the expression level of the gene sequence. The initial expression level of transcription factors is related to the growth state of cells. Therefore, the present disclosure combines the global resource regulation mechanism of the cell and the gene element to regulate the expression level of the gene. When the cells are nutrient-enriched, the growth rate of the cells is fast, and a large amount of cell resources are used to generate ribosomes. The background expression (transcription) level of transcription factors with constitutive transcriptional activity is low, and they can only be in an inactive state. The gene element is arranged upstream of the gene to be regulated, and the two are coupled, so that the expression level of the gene is also reduced. When cells are nutrient deficient, the growth rate of cells is slow, and a large amount of cell resources are allocated to produce proteins related to metabolism. The background expression (transcription) level of transcription factors with constitutive transcriptional activity is high, and the transcription factors are in a state of self-activation. At this time, the downstream genes to be regulated are affected by transcription factors, and their expression levels also increase.

The gene element is coupled with the growth rate of the cell, and through the change of the cell growth rate, the expression degree of the exogenous gene can be adjusted adaptively, thereby realizing automatic and programmable dynamic regulation of metabolism. The present disclosure involves very few genes to be modified, which are not directly connected to the metabolic network of cells, have good orthogonality, avoid unnecessary metabolic interference, and do not require a large amount of genetic modification of chassis cells.

In some embodiments, the transcription factor sequence is configured to express a transcription factor that is constitutively transcriptionally active, such that the transcription factor is transcriptionally active in the absence of an inducer.

Wherein, the term "constitutive transcriptionally active transcription factor" as used herein refers to a transcription factor obtained after being induced by an inducer or after binding with a specific molecule to change its conformation. In some cases, it has transcriptional activity or aggregates to form a multimer with transcriptional activity.

Since the transcription factor obtained by translation is in a designed conformation, it has constitutive transcriptional activity, and it also has transcriptional activity without the need of an inducer. Therefore, the transcription factor can interact with the promoter directly or after polymerization, and control the expression (transcription) of the downstream gene of the transcription factor sequence without the participation of the inducer. Suitable for expressing foreign proteins or enzymes with specific activities for the production of non-protein products. The present disclosure utilizes the modified activator to solve the problem of cost consumption of the inducer in the process of using the gene element, and the whole process occurs automatically and does not depend on the medium components.

In some embodiments, the transcription factor sequence is configured to express an AraC/xylS family protein having an N-terminal L-arabinose-bound conformation.

In the natural state, the transcription factor AraC protein in the endogenous L-arabinose response pathway of E. coli cells needs to bind L-arabinose and become activated AraC·L-ara to initiate the transcription of the Pbad promoter. Through the analysis of the crystal structure of the AraC/xylS family proteins, it is understood that the N-terminal domain of the AraC/xylS family proteins is responsible for binding with L-arabinose to change the conformation, and the two form a dimer to have transcriptional activity. The mutation of the sequence can realize the conformational transformation of the N-terminal domain and achieve constitutive transcriptional activity.

In some embodiments, the transcription factor comprises an AraC protein, rhaS protein, or rhaR protein with constitutive transcriptional activity. The promoter and activator protein used in the gene element can also be a combination of other activators and their promoters, including activators and corresponding promoters within the AraC/XylS family. The AraC, rhaS and rhaR proteins with constitutive transcriptional activity are variants of the wild-type AraC, rhaS and rhaR proteins in the AraC/xylS family by amino acid substitutions in the N- or C-terminal sequences. Variants of the AraC, rhaS and rhaR proteins can be freely selected according to the desired transcriptional strength.

In some embodiments, the transcription factor is AraC-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 1.

In some embodiments, the transcription factor is AraC-2 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 2.

In some embodiments, the transcription factor is AraC-3 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 3.

In some embodiments, the transcription factor is AraC-4 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 4.

In some embodiments, the transcription factor is AraC-5 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 5.

In some embodiments, the transcription factor is rhaS-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 6.

In some embodiments, the transcription factor is rhaR-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No. 7.

In some embodiments, the promoter sequence is configured as a wild-type Pbad promoter sequence or a wild-type Prhas promoter sequence.

In some embodiments, the promoter sequence is configured as the Pbad-1 promoter sequence after deleting the Crp binding site upstream of the wild-type Pbad promoter sequence, and the nucleotide sequence of the Pbad-1 promoter sequence is SEQ ID No.8.

In some embodiments, the promoter sequence is configured as the Prhas-1 promoter after deletion of the Crp binding site upstream of the wild-type Prhas promoter sequence, and the nucleotide sequence of the Prhas-1 promoter sequence is SEQ ID No. .9.

In some embodiments, the genetic element comprises a Pbad promoter and an AraC-1 protein, and the nucleotide sequence of the genetic element is SEQ ID No. 10.

In some embodiments, the genetic element is configured to regulate the expression of a gene or gene cluster.

In some embodiments, a terminator is also included, and the terminator is disposed upstream or downstream of the genetic element to suppress transcriptional interference.

In some embodiments, a fluorescent reporter gene is also included, and the fluorescent reporter gene is configured in the downstream sequence of the gene element for characterizing the expression level of the gene or gene cluster. The mVenus fluorescent reporter gene is placed downstream to characterize the expression levels of dynamically regulated genes or gene clusters.

The present disclosure also provides a method for using a gene element, comprising: culturing cells equipped with the gene element in a culture medium; when the concentration OD600 of the cells exceeds 0.2, collecting the cells and inoculating the diluted cells into a new culture Continue to cultivate in the base.

Streak the constructed strains equipped with genetic elements in a suitable culture plate, pick a moderate-sized single clone into LB medium, cultivate at 37°C for at least 3 hours, until its OD600 exceeds 0.2, collect cells by centrifugation, and use MOPS Resuspend and wash the cells with buffer solution, inoculate them into fresh SOB medium, control the OD600 concentration to be below 0.1, and shake culture at 37°C. Control the OD600 of the cells to always be lower than 0.2, and transfer when the OD is close to 0.2. Follow 1: The ratio of 100 to 500 was transferred to freshly preheated SOB medium, and when the OD600 reached 0.2 again, it was transferred to freshly preheated SOB medium at a ratio of 1:50 to 1000.

In some embodiments, the cells with this control circuit can be directly transferred into a nutrient-rich medium for one-step culture, and the medium can be commonly used mediums such as LB, SOB, YT, TB, RDM, and the like. In the early stage of growth, it is in a state of rapid growth without being restricted by nutrients in the environment, and the expression level of transcription factors in this gene element is low and in an inactive state. With the growth of cells and the increase of cell density, some nutrients are consumed, which limits the growth of cells. The growth rate of cells begins to slow down and enters the product production stage. The expression level of transcription factors is high, and the self-activation state is automatically turned on. The expression levels of downstream genes to which the transcription factor expression is coupled are also increased.

The present disclosure also provides another method for using a gene element, comprising: culturing cells equipped with the gene element in a medium; when the concentration OD600 of the cells exceeds 0.2, collecting the cells and inoculating the diluted cells into new cells Culture in the medium; when the cell concentration OD600 is close to 0.2, the new medium is supplemented with the precursor substances of the exogenous cell products and then the culture is continued.

Streak the constructed strains equipped with genetic elements in a suitable culture plate, pick a moderate-sized single clone into LB medium, cultivate at 37°C for at least 3 hours, until its OD600 exceeds 0.2, collect cells by centrifugation, and use MOPS The cells were resuspended and washed with buffer solution, inoculated into fresh RDM medium (glucose as carbon source), the OD600 concentration was controlled below 0.02, and the cells were shaken at 37 °C to culture, control the OD600 of the cells to be always lower than 0.2, and proceed when the OD600 was close to 0.2. Transfer, transfer to fresh RDM medium at a ratio of 1:100-500, transfer cells to fresh pre-warmed medium when the cell reaches 0.2 again, and prepare for feeding when the cell growth concentration OD600 is close to 0.2. The feed medium adopts MOPS medium (glucose as carbon source), and then continues to culture. Since the feed medium lacks nutrients such as amino acids that cells can directly utilize, the cell growth rate decreases and transcription factors are in an activated state.

The method of using the gene element can be used for fed-batch fermentation. In the cell growth stage, the cell grows at a high speed, the expression level of the transcription factor is low, and the cell is in an inactive state. When entering the production stage of cell products, the fed-batch medium is configured to maintain the cells in a low-speed growth state. At this time, the transcription factors are highly expressed and activated, and the downstream genes coupled with them are also expressed at a high level. In contrast, fed-feed fermentation can supplement a certain amount of exogenous product precursors during the product production stage of fermentation (the stage of low cell growth rate), which can increase the yield per unit volume and provide cells with a certain amount of nutrients for growth. components to extend the duration of the product production phase to further enhance the expression of foreign gene products.

In some embodiments, genetic elements are deployed upstream of the sequence of the gene or gene cluster to be regulated by traceless assembly. The gene or gene cluster to be regulated is inserted downstream of the genetic element by standard seamless assembly methods.

In some embodiments, the genetic elements are deployed in the genome or plasmid of the cell by genetic assembly. Using standard gene assembly methods, such as type II-S restriction enzyme-T4 ligase seamless assembly method, Gibson assembly method or DNA chemical synthesis method to obtain gene elements, which can be loaded into plasmids by standard assembly methods or Use directly knock-in cells into the genome.

In some embodiments, the sequence of the gene element, the sequence consisting of the same promoter sequence as the gene element and the sequence of the gene or gene cluster to be regulated in tandem are configured and used in the same cell.

In some embodiments, the cells are configured as one of E. coli cells, Bacillus subtilis, Lactobacillus, Streptomyces, yeast, Corynebacterium, animal cells. The invention creates a genetic background independent of cells and is suitable for various common cell chassis.

In some embodiments, the medium is configured as one or more of LB medium, SOB medium, YT medium, TB medium, RDM medium.

In an embodiment of the present disclosure, a schematic structural diagram of a gene element is shown in FIG. 1 , and the gene element includes a transcription factor-specific promoter sequence 1 and a transcription factor sequence 2 connected in series with each other. Downstream of the transcription factor sequence is the gene sequence 3 of the gene to be regulated.

In an embodiment of the present disclosure, according to the relationship between the cell growth rate and the expression level of the gene to be regulated, as shown in FIG. 2 , in the rapid growth stage, the expression level of the regulated gene is very low (L1), and as the cell growth rate becomes slower , the expression level of the regulated gene increases rapidly, and the gene circuit is in an activated state (L2).

In one embodiment of the present disclosure, the constructed strains configured with genetic elements are streaked in a suitable culture plate, and single clones of moderate size are picked into LB medium, and cultured at 37°C for at least three hours, until the When the OD600 exceeds 0.2, the cells are collected by centrifugation. The cells are resuspended and washed with MOPS buffer, inoculated into fresh SOB medium, and the OD600 concentration is controlled below 0.1. When it is close to 0.2, transfer to freshly preheated SOB medium at a ratio of 1:100 to 500. When OD600 reaches 0.2 again, transfer to freshly preheated SOB medium at a ratio of 1:50 to 1000. At the same time, samples were taken regularly, and the expression level and OD600 of the cell reporter gene were determined, which were used to characterize the expression intensity of exogenous and exogenous genes in the cell and calculate the growth rate of the cell. The experimental results are shown in Figure 3. After the cell growth rate dropped to 1.0/h, the gene expression level began to rise. When the OD600 approached 0.2, the cell growth rate began to decline rapidly. At this time, the dynamic regulation gene circuit entered an activated state, and the reporter gene The signal rises rapidly, at which time the cell growth rate is maintained at a lower growth rate. After about 8h, the growth of cells basically entered a state of stagnation, and the exogenous expression of cells reached the highest value at this time.

In one embodiment of the present disclosure, the constructed strains configured with genetic elements are streaked in a suitable culture plate, and single clones of moderate size are picked into LB medium, and cultured at 37°C for at least three hours, until the When the OD600 exceeds 0.2, the cells are collected by centrifugation, resuspended and washed with MOPS buffer, and inoculated into fresh RDM medium (glucose as carbon source), and the OD600 concentration is controlled below 0.02. Always lower than 0.2, transfer when the OD600 is close to 0.2, transfer to fresh RDM medium at a ratio of 1:100-500, and transfer cells to fresh prewarmed medium when the cell growth concentration reaches 0.2 again. When the OD600 is close to 0.2, it is ready to be fed. The feed medium uses MOPS medium (glucose as a carbon source) and continues to culture. Because the feed medium lacks nutrients such as amino acids that cells can directly utilize, the cell growth rate decreases and the dynamic The regulatory circuit was turned on, and during the whole process, the cell density and the expression intensity of the reporter gene were measured. The experimental results are shown in Figure 4.

As shown in Figure 4, in the process of fed-feed fermentation, when the cell growth rate decreased and entered the cell product production stage, under the premise of using feed medium with different formulas, the expression level of the gene to be regulated was relative to the expression in the rapid growth stage. Level difference (gene expression level at production stage/gene expression level at growth stage).

Although the present invention has been described with reference to the current preferred embodiments, those skilled in the art should understand that the above preferred embodiments are only used to illustrate the present invention, not to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the scope of the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

A gene element for regulating gene expression, characterized in that it has:

a transcription factor sequence, the expression of which is configured to be coupled to the expression of the gene;

A promoter sequence configured upstream of the transcription factor sequence for initiating expression of sequences downstream of the promoter.
The genetic element of claim 1, wherein the transcription factor sequence is configured to express a transcription factor with constitutive transcriptional activity such that the transcription factor is transcriptionally active in the absence of an inducer.
The genetic element of claim 2, wherein the transcription factor sequence is configured to express an AraC/xylS family protein having an N-terminal conformation bound to L-arabinose.
The gene element according to claim 3, wherein the transcription factor is AraC-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.1.
The gene element according to claim 3, wherein the transcription factor is AraC-2 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.2.
The gene element according to claim 3, wherein the transcription factor is AraC-3 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.3.
The gene element according to claim 3, wherein the transcription factor is AraC-4 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.4.
The gene element according to claim 3, wherein the transcription factor is AraC-5 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.5.
The gene element according to claim 3, wherein the transcription factor is rhaS-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.6.
The gene element according to claim 3, wherein the transcription factor is rhaR-1 protein, and the nucleotide sequence of the transcription factor is SEQ ID No.7.
The genetic element of claim 3, wherein the promoter sequence is configured as a wild-type Pbad promoter sequence or a wild-type Prhas promoter sequence.
The gene element according to claim 11, wherein the promoter sequence is configured to delete the Pbad-1 promoter sequence after the Crp binding site upstream of the wild-type Pbad promoter sequence, the Pbad- 1 The nucleotide sequence of the promoter sequence is SEQ ID No. 8.
The genetic element of claim 12, wherein the promoter sequence is configured as a Prhas-1 promoter after deletion of a Crp binding site upstream of a wild-type Prhas promoter sequence, the Prhas-1 The nucleotide sequence of the promoter sequence is SEQ ID No. 9.
The gene element according to any one of claims 1 to 13, wherein the gene element comprises a Pbad promoter and an AraC-1 protein, and the nucleotide sequence of the gene element is SEQ ID No. 10.
The genetic element of any one of claims 1 to 13, wherein the genetic element is configured to regulate the expression of a gene or gene cluster.
The gene element according to claim 15, further comprising a terminator, the terminator is configured in the upstream or downstream sequence of the gene element for suppressing transcriptional interference.
The gene element according to claim 16, further comprising a fluorescent reporter gene, wherein the fluorescent reporter gene is arranged in the downstream sequence of the gene element, and is used to characterize the expression level of the gene or gene cluster.
A method of using a gene element, characterized in that the method comprises:

culturing cells equipped with the genetic element in a culture medium;

When the concentration OD600 of the cells exceeds 0.2, the cells are collected and the diluted cells are inoculated into a new medium to continue culturing.
A method of using a gene element, comprising:

culturing cells equipped with the genetic element in a culture medium;

When the concentration OD600 of the cells exceeds 0.2, collect the cells and inoculate the diluted cells into a new medium for culture;

When the OD600 of the cell concentration is close to 0.2, the new medium is supplemented with the precursor substances of the exogenous cell products and the culture is continued.
The method for using a gene element according to claim 18 or 19, wherein the gene element is configured to be used upstream of the sequence of the gene or gene cluster to be regulated by a traceless assembly method.
The method of using a gene element according to claim 18 or 19, wherein the gene element is configured to be used in a genome or a plasmid of a cell by a gene assembly method.
The method for using a gene element according to claim 18 or 19, wherein the sequence of the gene element is composed of the same promoter sequence as the gene element and the sequence of the gene or gene cluster to be regulated in series. Sequence configurations are used within the same cell.
The method for using a gene element according to claim 18 or 19, wherein the cell is configured as one of Escherichia coli cells, Bacillus subtilis, lactic acid bacteria, Streptomyces, yeast, Corynebacterium, and animal cells .
The method for using a gene element according to claim 18 or 19, wherein the medium is configured as one of LB medium, SOB medium, YT medium, TB medium, and RDM medium or variety.