Method for regulating and controlling expression intensity of prpB gene promoter

Abstract

The invention provides a method for regulating and controlling the expression intensity of a prpB gene promoter, specifically, betaine is added into an expression system containing the prpB gene promoter, and the expression intensity of the prpB gene promoter can be regulated and controlled to be improved in the logarithmic phase and / or reduced in the stable phase of microbial growth. By utilizing the method, the expression level of a target gene in an expression system can be accurately regulated and controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a method for regulating the expression intensity of a prpB gene promoter by adding betaine. Background Art

[0002] The promoter is a DNA sequence that RNA polymerase specifically recognizes and binds to. It is a component of the gene and controls the start time of gene transcription and the degree of transcription expression like a "switch". The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can guide the holoenzyme (Holoenzyme) to correctly bind to the template, activate RNA polymerase, and start gene transcription. The promoter itself cannot directly control gene expression, but controls structural gene expression by binding to transcription factors.

[0003] Currently, commonly used promoter types include constitutive promoters (promoters that can activate gene expression in all cells) and inducible promoters (specific inducible promoters can activate the transcriptional expression of downstream structural genes only under the stimulation of certain physical signals, chemical signals or small molecule signals). The regulation of constitutive promoters is not affected by external conditions, and the expression of the activated genes is continuous, but it does not show time specificity, cannot be artificially regulated, and is not operable in practical applications; while inducible promoters can artificially regulate the expression of structural genes through special external signals. At present, commonly used induction signals include chemical substances such as IPTG and lactose, which face problems such as high costs in actual production processes.

[0004] Betaine (Betaine, molecular formula is C5H 12 NO2) has multiple physiological functions such as improving the tolerance of cells to the osmotic pressure of the extracellular environment, providing methyl donors for the synthesis of target products, and protecting the activity of key enzymes in organisms, and is widely used in microbial fermentation production processes. CN106636023A announced that the expression intensity of the ZWF gene promoter was enhanced by 0.2-0.98 times by adding betaine to the expression system or fermentation system. In addition, the induction function of betaine has not yet been recognized, developed and utilized. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a method for regulating the expression intensity of the prpB gene promoter, specifically by adding betaine to the microbial expression system to achieve the purpose of increasing and / or decreasing the expression intensity of the prpB gene promoter.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for regulating the expression intensity of a prpB gene promoter, wherein betaine is added to an expression system containing a prpB gene promoter, thereby regulating the expression intensity of the prpB gene promoter to increase during the logarithmic phase of microbial growth and / or decrease during the stable phase.

[0008] As described above in the method provided by the present invention in the first aspect, the expression intensity of the prpB gene promoter is increased by 160%-180% during the logarithmic phase of microbial growth.

[0009] As in the method provided by the present invention in the first aspect above, the expression intensity of the prpB gene promoter is reduced by 70%-80% during the stable period of microbial growth.

[0010] In a second aspect, the present invention provides a method for regulating the expression level of a target gene, the method comprising adding betaine to an expression system containing the target gene and its upstream prpB gene promoter, thereby regulating the expression level of the target gene to increase during the logarithmic phase of microbial growth and / or decrease during the stable phase.

[0011] The beneficial effects of the present invention are as follows:

[0012] In the present invention, betaine added to the microbial fermentation system is different from a common methyl donor, but is used as a new inducer to regulate the expression intensity of the prpB gene promoter in the expression system to significantly increase in the logarithmic phase and / or significantly decrease in the stable phase; thus, the method can be used to achieve precise regulation of the expression level of the target gene in the expression system. Realizing this precise regulation has the following advantages or effects:

[0013] Rapid response: The rapid increase in promoter expression intensity enables cells to respond quickly to external stimuli or internal needs and initiate the expression of related genes.

[0014] Precise regulation: The subsequent reduction in expression intensity helps avoid excessive consumption of resources and unnecessary gene expression, achieving precise regulation of gene expression.

[0015] Adapting to environmental changes: This regulatory mode enables cells to better adapt to changing environmental conditions and maintain the stability of the internal environment.

[0016] Avoid negative effects: High expression of certain genes may produce negative effects, which can be avoided by reducing the expression intensity in a timely manner through precise regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Comparison of prpB gene transcription levels under the action of betaine in Example 1.

[0018] Figure 2: Comparison of strain growth curves under the action of betaine in Example 1. DETAILED DESCRIPTION

[0019] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0020] In a first aspect, the present invention provides a method for regulating the expression intensity of a prpB gene promoter, wherein betaine is added to an expression system containing a prpB gene promoter, thereby regulating the expression intensity of the prpB gene promoter to increase during the logarithmic phase of microbial growth and / or decrease during the stable phase.

[0021] According to the method provided by the present invention, the expression intensity of the prpB gene promoter is regulated to increase during the logarithmic phase of microbial growth, for example, by 100% or more, 120% or more, 140% or more, 160% or more, 180% or more, or 200% or more.

[0022] According to a preferred embodiment of the present invention, the expression intensity of the prpB gene promoter is increased by 160%-180% during the logarithmic phase of microbial growth.

[0023] According to the method provided by the present invention, the expression intensity of the prpB gene promoter is regulated to decrease during the stable growth period of the microorganism, for example, by 40%, 50%, 60%, 70%, 80%, 90%, 100%.

[0024] According to a preferred embodiment of the present invention, the expression intensity of the prpB gene promoter is reduced by 70%-80% during the stable period of microbial growth.

[0025] According to a preferred embodiment of the present invention, the amount of betaine added is a working concentration of 1 g / L in the fermentation system.

[0026] According to the present invention, the expression system can be a common bacterial expression system, for example, an Escherichia coli expression system or a Bacillus subtilis expression system.

[0027] According to a preferred embodiment of the present invention, the expression system is an Escherichia coli expression system.

[0028] According to a preferred embodiment of the present invention, in the Escherichia coli expression system with the addition of betaine, the expression intensity of the prpB gene promoter increases from the beginning of fermentation to 12h, especially by 160%-180% at 8-12h; and decreases after 16h of fermentation, especially by 70%-80% at 16-20h.

[0029] According to a preferred embodiment of the present invention, the nucleotide sequence of the prpB gene promoter is shown as SEQ ID No.1.

[0030] According to the present invention, the prpB gene encodes methylisocitrate lyase, which has the ability to lyse 2-methylisocitrate into pyruvate and succinate.

[0031] In a second aspect, the present invention provides a method for regulating the expression level of a target gene, the method comprising adding betaine to an expression system containing the target gene and its upstream prpB gene promoter, thereby regulating the expression level of the target gene to increase during the logarithmic phase of microbial growth and / or decrease during the stable phase.

[0032] The term "logarithmic phase" or "logarithmic growth phase", also called exponential growth phase, refers to a period of time when the number of microbial cells increases exponentially after the delay period. During this period, the nutrition is sufficient, the cells grow rapidly, the number of viable bacteria increases at a constant geometric progression, and the logarithm of the number of bacteria on the growth curve rises linearly and reaches a peak state. The morphology, staining, physiological activity, etc. of the bacteria are relatively typical and sensitive to the effects of environmental factors.

[0033] The term "stable phase" or "stable growth phase" refers to the phase after the logarithmic phase and before the decay phase, during the growth of the microbial population, when the number of newly propagated cells and the number of dead cells are basically in a stable equilibrium, the number of live cells reaches a dynamic equilibrium, and the growth rate of the population is equal to zero. During this phase, nutrients are gradually depleted and harmful metabolites accumulate, resulting in a slowdown in the reproduction rate of microorganisms and a slow increase in the number of deaths, but the total number of live bacteria remains relatively stable and reaches the highest level.

[0034] The present invention will be described in more detail below through specific examples. The specific operation means of molecular biology, genetic engineering, etc. involved in the embodiments of the present invention can be implemented according to technical manuals, textbooks or literature reports easily available to those skilled in the art, and it is not necessary to describe the operation process in detail here.

[0035] Embodiment 1:

[0036] This embodiment provides a method for regulating the expression intensity of the prpB gene promoter in an Escherichia coli expression system, comprising the following experimental steps:

[0037] 1) Take a loop of E. coli MG1655 bacterial solution from the glycerol tube with an inoculating loop and place it on a non-antibiotic activated slant medium, streak it densely, and culture it at 37°C for 12 hours (first generation slant);

[0038] 2) Take a loopful of bacteria from the first generation slant and streak it onto another slant, and culture it at 37°C for 8 hours (second generation slant);

[0039] 3) Take a loopful of bacteria from the second generation slant and transfer it to 50 mL of seed medium. Culture at 37°C and 200 rpm for 8 h.

[0040] 4) Inoculate 20% of the inoculum into 30 mL of fermentation medium, culture at 37°C, 200 rpm, and adjust the pH to 6.7-7.0 with ammonia water. No betaine was added to the control medium, and betaine was added to the experimental medium at a final concentration of 1 g / L. Samples were taken at 8 h, 12 h, 16 h, and 20 h of fermentation to determine the transcription amount, and the transcription level of the prpB gene, a dynamic significant difference gene under the action of betaine, was verified.

[0041] 5) Culture medium formula:

[0042] Activated slant medium (g / L): glucose 1, peptone 10, beef extract 5, yeast powder 5, NaCl 2.5, agar powder 20, adjust pH to 7.0-7.2, sterilize at 121℃ for 20min.

[0043] Seed culture medium (g / L): sucrose 25, yeast powder 10, peptone 6, KH2PO4 1.2, MgSO4·7H2O 0.5, FeSO4·7H2O 0.01, MnSO4·3H2O 0.01, V B1 1.3mg / L,V H 0.3mg / L, adjust pH to 7.0-7.2, add 1-2 drops of defoaming agent, sterilize at 115℃ for 15min.

[0044] Fermentation medium (g / L): glucose 30, yeast powder 2, peptone 4, sodium citrate 1, KH2PO42, MgSO4·7H2O0.7, FeSO4·7H2O 0.1, MnSO4·3H2O 0.1, V B1 0.0008, V H 0.0002, adjust pH to 7.0-7.2, and sterilize at 115℃ for 15min.

[0045] Experimental results:

[0046] Comparison of prpB gene promoter transcription levels at different time periods Figure 1As shown. At 8h and 12h of fermentation, the transcription level of the prpB gene promoter in the experimental group (1g / L betaine added) increased by 160% and 180% respectively compared with that in the control group (without betaine added); at 16h and 20h of fermentation, the transcription level of the prpB gene promoter in the experimental group (1g / L betaine added) decreased by 80% and 70% respectively compared with that in the control group (without betaine added). The results show that the addition of betaine to the microbial expression system can significantly regulate the expression intensity of the prpB gene promoter, and the regulation includes positive regulation and negative regulation, which is specifically manifested in that the expression intensity of the prpB gene promoter can be significantly enhanced in the logarithmic growth period (especially 8-12h) and / or significantly decreased in the stable period (especially 16-20h). In addition, according to the strain OD 600 Growth curve Figure 2 As shown, there was no significant difference in the growth of strains between the experimental group and the control group.

[0047] Embodiment 2:

[0048] This embodiment provides a method for regulating the expression level of a target gene in an expression system containing a prpB gene promoter and a green fluorescent protein gene gfp, comprising the following experimental steps:

[0049] 1) Amplification of prpB gene promoter and gfp gene and construction of overlapping fragments:

[0050] The amplification primers prpB-S and prpB-A were designed based on the promoter sequence of the prpB gene in the E. coli MG1655 genome; the amplification primers gfp-S and gfp-A were designed based on the gfp gene sequence. The primers prpB-A and gfp-S contain a 20 bp overlapping sequence, and the 5' ends of the primers prpB-S and gfp-A contain homologous sequences upstream and downstream of the vector to be cloned. The primers and promoter sequences involved are as follows:

[0051]

[0052] The high-fidelity enzyme PrimeSTAR HSDNA Polymerase was used to amplify the prpB gene promoter and the gfp gene, respectively. The PCR products were recovered using the OMEGA PCR product purification kit of Guangzhou Feiyang Biotechnology Co., Ltd. to obtain the PprpB and gfp fragments.

[0053] The PCR system is as follows:

[0054]

[0055] PrimeSTAR HSDNA Polymerase was then used toprpB The fragment and gfp fragment were overlapped by PCR amplification, and the P prpB -gfp is for standby use.

[0056] The overlapping PCR system is as follows:

[0057]

[0058] 2) Construction of vector pSTV28-P prpB -gfp:

[0059] The vector pSTV28 and the overlapping fragment P were cloned using the ClonExpress-II One Step Cloning Kit. prpB -gfp for recombination ligation.

[0060] The recombinant connection system is as follows:

[0061]

[0062]

[0063] 3) Transformation of recombination ligation products:

[0064] ① Place the recombinant ligation product in a 37°C water bath for 30 minutes, then place it in an ice bath for 5 minutes, add it to the competent E. coli DH5α medium, mix it with a pipette, and continue to place it in an ice bath for 20 minutes;

[0065] ② Place the EP tube in a 42℃ water bath and heat shock for 60s;

[0066] ③Incubate on ice for 2 minutes, add 800 μL of SOC recovery solution, and incubate at 37°C, 200 rpm for 1 hour;

[0067] ④ Centrifuge at 8000rpm for 2min, retain 100μL of liquid, resuspend the bacteria, spread on LB plates containing ampicillin resistance, and culture at 37℃ overnight.

[0068] 4) Verification of positive clones:

[0069] The colony PCR system is as follows:

[0070]

[0071] Use tweezers to hold the toothpick in the clean bench, quickly pass the tip of the toothpick through the flame 2-3 times, and accurately pick a single colony on the plate with a single colony with the tip of the toothpick. First, gently draw a line on the plate with grid lines, and then put the toothpick into the corresponding PCR tube and rinse it several times. After electrophoresis verification, the positive strain was obtained, and the strain with the correct number on the plate was inoculated into the LB shake tube, cultured at 37℃ overnight, and stored in the glycerol tube at -80℃, named E.coliDH5α-pSTV28-P prpB -gfp.

[0072] Determination of E.coli DH5α-pSTV28-P under the condition of exogenous addition of betaine prpB The fluorescence intensity of the -gfp strain is shown in Table 1. The results show that when betaine is added to the fermentation system at a final concentration of 1 g / L, E. coli DH5α-pSTV28-P prpB Compared with the control group (without betaine), the fluorescence intensity of the -gfp strain increased significantly at 8-12h of fermentation, indicating that the expression level of the gfp gene increased significantly; the fluorescence intensity decreased significantly at 16-20h of fermentation, indicating that the expression level of the gfp gene decreased significantly. This experiment further confirmed the precise regulation of betaine on the expression level of the target gene controlled by the prpB gene promoter.

[0073]

[0074] Table 1 Effect of betaine on E.coli DH5α-pSTV28-P prpB Effect of -GFP strain on fluorescence intensity

[0075]

[0076] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for regulating the expression intensity of the prpB gene promoter, characterized in that: The method comprises adding betaine to an expression system containing a prpB gene promoter, and can regulate the expression intensity of the prpB gene promoter to increase in the logarithmic phase of microbial growth and / or decrease in the stable phase.

2. The method according to claim 1, characterized in that The expression intensity of the prpB gene promoter is increased by 160%-180% in the logarithmic phase of microbial growth.

3. The method according to claim 1, characterized in that The expression intensity of the prpB gene promoter is reduced by 70%-80% during the stable growth period of the microorganism.

4. The method according to claim 1, characterized in that The expression system is an Escherichia coli expression system.

5. The method according to claim 4, characterized in that The expression intensity of the prpB gene promoter increased by 160%-180% during fermentation for 8-12 hours, and decreased by 70%-80% during fermentation for 16-20 hours.

6. The method according to claim 1, characterized in that The amount of betaine added is 1 g / L at a working concentration in the fermentation system.

7. The method according to claim 1, characterized in that The nucleotide sequence of the prpB gene promoter is shown in SEQ ID No.

1.

8. A method for regulating the expression level of a target gene, the method comprising adding betaine to an expression system containing the target gene and its upstream prpB gene promoter, so as to regulate the expression level of the target gene to increase during the logarithmic phase of microbial growth and / or decrease during the stable phase.

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