A constitutive promoter P256 and its activity identification method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to a constitutive promoter P256 and its activity identification method and application. The constitutive promoter P256 is derived from the Arctic psychrophilic bacterium Pseudoalteromonas fuliginea BSW20308. After amplification, it is cloned into the upstream of the green fluorescent protein coding region of the pRU1701 plasmid after enzyme digestion using a seamless assembly kit to obtain a recombinant plasmid. The recombinant plasmid and the blank vector pRU1701 plasmid are then transformed into Escherichia coli DE3, cultured in LB medium, and samples are taken after inoculation to detect their fluorescence value and OD. 600 The activity of the constitutive promoter P256 was identified using unit fluorescence intensity. The constitutive promoter P256 of the present invention has the potential to be applied to other different types of prokaryotic bacteria, including special extreme environment microorganisms, and can be used to increase the expression of exogenous proteins.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a constitutive promoter P256 and an activity identification method and application thereof. Background Art

[0002] Promoters are the binding sites for RNA polymerase and transcription factors, typically located upstream of genes. They play a crucial role in regulating anabolism and metabolism. They are primarily classified into three types: RNA polymerase I, RNA polymerase II, and RNA polymerase III. The promoters of conventional genes in cells are almost all type II promoters, characterized by their unlimited transcriptional length. Type II promoters include constitutive and inducible promoters. Inducible promoters require the addition of specific substances to stimulate or inhibit their activity. Constitutive promoters are the most commonly used in expression systems. These promoters maintain a certain level of expression activity in most cells, but different constitutive promoters can exhibit varying levels of expression in different cells.

[0003] The Antarctic and Arctic possess some of the most unique geographical and climatic conditions in the world. Microorganisms surviving these extreme environments have developed unique adaptation mechanisms, often exhibiting characteristics such as cold-loving, salt-tolerant, and pressure-tolerant traits, thus possessing significant scientific value. The development and utilization of polar microbial resources has shown promising application prospects, with numerous high-quality functional genes already identified. However, reports on constitutive promoters are few and far outstrip the growing number of genes. Summary of the Invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide a constitutive promoter P256 and its activity identification method and application. The constitutive promoter P256 is derived from the Arctic psychrophilic bacterium Pseudoalteromonas fuliginea BSW20308. After amplification, it is cloned into the upstream of the green fluorescent protein coding region of the pRU1701 plasmid after enzyme digestion using a seamless assembly kit to obtain a recombinant plasmid; then the recombinant plasmid and the blank vector pRU1701 plasmid are respectively transformed into Escherichia coli E. coli DE3 and cultured in LB medium. After inoculation, samples are taken to detect their fluorescence value and OD 600 The activity of the constitutive promoter P256 was identified using unit fluorescence intensity.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first object of the present invention is to provide a constitutive promoter P256, wherein the constitutive promoter P256 is derived from the Arctic psychrophilic bacterium Pseudoalteromonas fuliginea BSW20308, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0007] A second object of the present invention is to provide a method for identifying the activity of a constitutive promoter P256, comprising the following steps:

[0008] (1) The constitutive promoter P256 in the genome of the Arctic bacterium Pseudoalteromonas fuliginea BSW20308 was amplified by PCR using the primer pair to obtain the amplified product;

[0009] (2) cloning the amplified product obtained in step (1) into the upstream of the green fluorescent protein coding region of the pRU1701 plasmid after enzyme digestion using a seamless assembly kit to obtain a recombinant plasmid;

[0010] (3) The recombinant plasmid obtained in step (2) and the blank vector pRU1701 plasmid were transformed into E. coli DE3, cultured in LB medium, and samples were taken after inoculation to detect the fluorescence value and OD 600 The activity of the constitutive promoter P256 was identified using unit fluorescence intensity.

[0011] In one embodiment of the present invention, in step (1), the primer pair is P-1 and P-2; wherein the nucleotide sequence of P-1 is shown as SEQ ID NO.2, and the nucleotide sequence of P-2 is shown as SEQ ID NO.3.

[0012] In one embodiment of the present invention, in step (1), during the PCR amplification process, the reaction conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 20 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, for a total of 35 cycles; and extension at 72°C for 5 min.

[0013] In one embodiment of the present invention, in step (2), the pRU1701 plasmid after enzyme digestion is the pRU1701 plasmid digested with SpeⅠ.

[0014] In one embodiment of the present invention, in step (3), the LB culture medium contains gentamicin.

[0015] In one embodiment of the present invention, in step (3), the fluorescence value of the sample is measured using a Fluoroskan Ascent FL automatic fluorescence chemiluminescence analyzer; the OD of the sample is measured using a light absorption microplate reader. 600 .

[0016] In one embodiment of the present invention, in step (3), when the fluorescence value is detected, the excitation wavelength is 485 nm and the emission wavelength is 538 nm.

[0017] In one embodiment of the present invention, in step (3), the fluorescence value is divided by the corresponding OD 600 is the unit fluorescence intensity. When the unit fluorescence intensity of the recombinant plasmid is higher than that of the blank vector pRU1701 plasmid, it proves that the activity of the constitutive promoter P256 is high; otherwise, it proves that the activity of the constitutive promoter P256 is low.

[0018] The third object of the present invention is to provide an application of a constitutive promoter P256, wherein the constitutive promoter P256 is used to be applied to other different types of prokaryotic bacteria to increase the expression level of exogenous proteins.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention identified a constitutive promoter P256 in the genome of the Arctic psychrophilic bacterium Pseudoalteromonas fuliginea BSW20308. By constructing a recombinant vector, the promoter remains active in Escherichia coli and does not require an inducer. It is a constitutive promoter with universal activity and has the potential to be applied to other different species of prokaryotic bacteria, including special extreme environment microorganisms. Furthermore, the constitutive promoter P256 can be used to increase the expression level of exogenous proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the recombinant plasmid constructed in Example 1;

[0022] Figure 2 The GFP unit fluorescence intensity of the recombinant plasmid and blank plasmid constructed in Example 1 at different times; *** represents P < 0.001. DETAILED DESCRIPTION

[0023] The present invention provides a constitutive promoter P256. The constitutive promoter P256 is derived from Arctic psychrophilic bacterium Pseudoalteromonas fuliginea BSW20308, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0024] The present invention provides a method for identifying the activity of a constitutive promoter P256, comprising the following steps:

[0025] (1) The constitutive promoter P256 in the genome of the Arctic bacterium Pseudoalteromonas fuliginea BSW20308 was amplified by PCR using the primer pair to obtain the amplified product;

[0026] (2) cloning the amplified product obtained in step (1) into the upstream of the green fluorescent protein coding region of the pRU1701 plasmid after enzyme digestion using a seamless assembly kit to obtain a recombinant plasmid;

[0027] (3) The recombinant plasmid obtained in step (2) and the blank vector pRU1701 plasmid were transformed into E. coli DE3, cultured in LB medium, and samples were taken after inoculation to detect the fluorescence value and OD 600 The activity of the constitutive promoter P256 was identified using unit fluorescence intensity.

[0028] In one embodiment of the present invention, in step (1), the primer pair is P-1 and P-2; wherein the nucleotide sequence of P-1 is shown as SEQ ID NO.2, and the nucleotide sequence of P-2 is shown as SEQ ID NO.3.

[0029] In one embodiment of the present invention, in step (1), during the PCR amplification process, the reaction conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 20 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, for a total of 35 cycles; and extension at 72°C for 5 min.

[0030] In one embodiment of the present invention, in step (2), the pRU1701 plasmid after enzyme digestion is the pRU1701 plasmid digested with SpeⅠ.

[0031] In one embodiment of the present invention, in step (3), the LB culture medium contains gentamicin.

[0032] In one embodiment of the present invention, in step (3), the fluorescence value of the sample is measured using a Fluoroskan Ascent FL automatic fluorescence chemiluminescence analyzer; the OD of the sample is measured using a light absorption microplate reader. 600 .

[0033] In one embodiment of the present invention, in step (3), when the fluorescence value is detected, the excitation wavelength is 485 nm and the emission wavelength is 538 nm.

[0034] In one embodiment of the present invention, in step (3), the fluorescence value is divided by the corresponding OD 600is the unit fluorescence intensity. When the unit fluorescence intensity of the recombinant plasmid is higher than that of the blank vector pRU1701 plasmid, it proves that the activity of the constitutive promoter P256 is high; otherwise, it proves that the activity of the constitutive promoter P256 is low.

[0035] The present invention provides an application of a constitutive promoter P256, wherein the constitutive promoter P256 is used for being applied to other different kinds of prokaryotic bacteria to increase the expression amount of exogenous proteins.

[0036] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] In the following examples, unless otherwise specified, all reagents used are commercially available reagents; and all detection means and methods used are conventional detection means and methods in the art.

[0038] The primer synthesis and gene sequencing used in the following examples were completed by Sangon Biotechnology (Shanghai) Co., Ltd. DE3 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Seamless assembly kit was purchased from Nanjing Novozymes Co., Ltd. Endonuclease SpeⅠ was purchased from Beijing New England Biotechnology Co., Ltd. LB medium and 2216E medium were purchased from BD Biosciences, USA; TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.2.0 and PrimeSTAR MaxPremix kit were purchased from Beijing Baoriyi Biotechnology Co., Ltd. Gentamycin was purchased from Sigma, USA; PCR product purification kit was purchased from Aisijin Biotechnology (Hangzhou) Co., Ltd.

[0039] Example 1

[0040] This example provides a method for identifying the activity of the constitutive promoter P256.

[0041] (1) Genome extraction: 4 ml of the Arctic bacterium Pseudoalteromonas fuliginea BSW20308 was cultured to an OD of 600 ≈1.0, the strain culture medium was 2216E (BD Biosciences, USA), the culture temperature was 25°C, the bacteria were collected by centrifugation, and the genome was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.2.0 (Baori Biotechnology Co., Ltd., Beijing, China).

[0042] (2) PCR amplification: The genome extracted in step (1) was used as a template and the primer pair P-1 / P-2 was used for PCR amplification. The system is shown in Table 1. The PCR reaction conditions were: 95°C pre-denaturation for 5 min, 95°C denaturation for 20 sec, 55°C annealing for 30 sec, and 72°C extension for 30 sec, for a total of 35 cycles; 72°C extension for 5 min. The amplified fragment was the constitutive promoter P256, with a length of 209 bp. The amplified product was purified and recovered using a PCR product purification kit (Aishengjin, Hangzhou, China).

[0043] Table 1 PCR reaction system

[0044] PCR reaction system 50μl system PrimeSTAR Max Premix 25 μl Genome 2 μl P-1 (SEQ ID NO. 2) 2 μl P-2 (SEQ ID NO. 3) 2 μl <![CDATA[ddH2O]]> Make up to 50 μl

[0045] Among them, PrimeSTAR Max Premix was purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.

[0046] P-1: 5'-TAGATAGAGAGAGAGAGAGATTAGCTATTGCATAAATACT-3';

[0047] P-2: 5'-ATTTTTTCTTCCTCCACTAGTTTTTACAACATTCCATTGT-3'.

[0048] (3) Enzyme digestion of plasmids: First, prepare the enzyme digestion system (50 μl): 2 μl of SpeⅠ (New England Biotechnology, Beijing, China), 5 μl of buffer, 2 μg of pRU1701 plasmid, and make up to 50 μl with sterile water. The prepared enzyme digestion system was placed in a 37°C water bath and allowed to stand for 1 h. After removal, the product was purified and recovered using a PCR product purification kit (Aixin, Hangzhou, China).

[0049] (4) Seamless assembly of recombinant plasmids - Prepare a seamless assembly reaction system (20 μl): 12 μl of pRU1701 plasmid digested in step (3), 4 μl of CEⅡ Buffer, 2 μl of amplified product, and 2 μl of seamless assembly enzyme (Novozymes, Nanjing, China). Place the prepared reaction system in a 37°C water bath, let it stand for 30 min, then remove it and cool it on ice for 3 min.

[0050] (5) Transformation of plasmids: 10 μl of the reaction solution obtained in step (4) and 10 μl of the pRU1701 plasmid were added to 100 μl of Escherichia coli competent cells DE3 (Quanshijin, Beijing, China). After standing on ice for 30 min, the cells were heat-shocked at 42°C for 90 s. After completion, the cells were placed on ice for 3 min. 1 ml of LB culture medium was added and the cells were cultured at 37°C for 1 h to restore the cell wall. The cells were then collected, part of the supernatant was discarded, and 100-200 μl of liquid was reserved. The cells were resuspended and spread on a plate containing gentamicin. The cells were cultured at 37°C overnight. The cells were picked the next day, and the activated bacterial solution containing the recombinant plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the recombinant plasmid pRU1701-P256 (as shown in Figure 2). Figure 1 Wherein, the final concentration of gentamicin in the solid culture medium or liquid culture medium using gentamicin is 10 mg / L.

[0051] (6) Measure the fluorescence intensity at different stages - activate DE3 containing pRU1701 plasmid and pRU1701-P256 plasmid respectively, and culture until OD 600 ≈0.6, inoculated into 30 ml of LB medium containing gentamicin at a 1% inoculum volume, with three replicates set up for each group. Samples were taken at 6 h, 9 h, 12 h, and 16 h after inoculation, with 1 ml sampled each time. After the cells were collected, they were washed three times with sterile water and temporarily stored at -80°C. After all the sampling was completed, 200 μl of sterile water was added to each sample to resuspend the cells. The fluorescence value of the sample was measured using a Fluoroskan Ascent FL fully automatic fluorescence chemiluminescence analyzer (Thermo Fisher Scientific, Waltham, USA), with the excitation wavelength set at 485 nm and the emission wavelength set at 538 nm. The OD of the sample was measured using an absorbance microplate reader. 600 Each sample was tested three times and the average of the three replicates was calculated. The fluorescence value was divided by the corresponding OD 600 , and obtain the unit fluorescence intensity. The experimental results are as follows Figure 2 As shown, from Figure 2 It was found that between 6 and 16 hours after inoculation, cells containing the constitutive promoter P256 had higher fluorescence intensity per cell, and this was significantly different from that of cells without the vector. The difference between the two reached its maximum around 12 hours after inoculation, indicating that the constitutive promoter P256 can significantly increase the expression of exogenous proteins (such as green fluorescent protein). It also demonstrated that the constitutive promoter P256 from Arctic psychrophilic bacteria still maintains high activity in E. coli.

[0052] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A constitutive promoter P256, characterized in that The constitutive promoter P256 is derived from the Arctic psychrophilic bacterium Pseudoalteromonas fuliginea BSW20308, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. A method for identifying the activity of the constitutive promoter P256 according to claim 1, characterized in that: The following steps are involved: (1) The constitutive promoter P256 in the genome of the Arctic bacterium Pseudoalteromonas fuliginea BSW20308 was amplified by PCR using the primer pair to obtain the amplified product; (2) cloning the amplified product obtained in step (1) into the upstream of the green fluorescent protein coding region of the pRU1701 plasmid after enzyme digestion using a seamless assembly kit to obtain a recombinant plasmid; (3) The recombinant plasmid obtained in step (2) and the blank vector pRU1701 plasmid were transformed into E. coli DE3, cultured in LB medium, and samples were taken after inoculation to detect the fluorescence value and OD 600 The activity of the constitutive promoter P256 was identified using unit fluorescence intensity.

3. The method for identifying the activity of a constitutive promoter P256 according to claim 2, wherein: In step (1), the primer pair is P-1 and P-2; wherein the nucleotide sequence of P-1 is shown as SEQ ID NO.2, and the nucleotide sequence of P-2 is shown as SEQ ID NO.

3.

4. The method for identifying the activity of a constitutive promoter P256 according to claim 2, wherein: In step (1), during the PCR amplification process, the reaction conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 20 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, for a total of 35 cycles; and extension at 72°C for 5 min.

5. The method for identifying the activity of the constitutive promoter P256 according to claim 2, wherein: In step (2), the pRU1701 plasmid after enzyme digestion is the pRU1701 plasmid digested with SpeⅠ.

6. The method for identifying the activity of the constitutive promoter P256 according to claim 2, wherein: In step (3), the LB culture medium contains gentamicin.

7. The method for identifying the activity of the constitutive promoter P256 according to claim 2, wherein: In step (3), the fluorescence value of the sample was measured using a Fluoroskan Ascent FL automatic fluorescence chemiluminescence analyzer; the OD of the sample was measured using an optical absorption microplate reader. 600 .

8. The method for identifying the activity of constitutive promoter P256 according to claim 7, wherein: In step (3), when the fluorescence value is detected, the excitation wavelength is 485 nm and the emission wavelength is 538 nm.

9. The method for identifying the activity of the constitutive promoter P256 according to claim 2, wherein: In step (3), the fluorescence value is divided by the corresponding OD 600 is the unit fluorescence intensity. When the unit fluorescence intensity of the recombinant plasmid is higher than that of the blank vector pRU1701 plasmid, it proves that the activity of the constitutive promoter P256 is high; otherwise, it proves that the activity of the constitutive promoter P256 is low.

10. A use of the constitutive promoter P256 according to claim 1, characterized in that: The constitutive promoter P256 is used to increase the expression level of exogenous proteins; The exogenous protein is green fluorescent protein.

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