Non-stable GFP escherichia coli heavy-labeled strain and application thereof

Abstract

The invention discloses a non-stable green fluorescent protein (GFP) escherichia coli heavy-labeled strain and application thereof, and belongs to the field of biological detection, the strain comprises a fusion expression unit of an rrnBP1 promoter and a non-stable green fluorescent protein gene, and the non-stable green fluorescent protein gene is a mutant formed by connecting an ASV degradation tag coding sequence to the C terminal of a gfpmut3b gene. A ribosome promoter rrnbp1 related to growth and metabolism of bacteria is utilized, strong fluorescence of GFP is transformed into non-stable fluorescence, a non-stable GFP strain is constructed, the non-stable GFP strain can respond to the growth and metabolism activity of the bacteria along with the rrnbp1 promoter, and low-metabolism drug-resistant bacteria are detected in a flora through the change of fluorescence intensity. The method solves the problem that the dormant drug-resistant bacteria cannot be detected in real time in the traditional method, has high sensitivity under the condition of adding sub-inhibition concentration antibiotics, and is suitable for clinical drug resistance monitoring and novel antibacterial drug development.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a non-stable GFP Escherichia coli heavy-labeled strain and application thereof. Background Art

[0002] Antimicrobial drugs treat infections by killing bacteria. However, bacteria, as a widespread class of organisms, can also acquire resistance to antimicrobial drugs in various forms, evading the danger of being killed. This resistance is called "bacterial resistance," and bacteria that acquire resistance are called "resistant bacteria." Bacterial resistance is a natural phenomenon that has been enhanced by humans. Bacterial resistance refers to the fact that after repeated exposure to drugs, bacteria's sensitivity to drugs decreases or even disappears, resulting in reduced or even ineffective efficacy of drugs against resistant bacteria. With the widespread use of antibiotics, bacterial resistance has become one of the issues that seriously affect human health. Persistent and chronic infections caused by resistant bacteria have become a major public health problem, among which the harm of resistant bacteria in a low-metabolic dormant state (such as persisters) is particularly prominent. Under the pressure of antibiotics, these bacteria enter a state of growth stagnation, and their metabolic activity plummets to less than one thousandth of their normal level. They not only evade conventional bactericidal mechanisms but also become the root cause of recurrent chronic infections.

[0003] Traditional bacterial detection and monitoring methods often rely on bacterial culture characteristics and biochemical reactions. These methods are cumbersome and time-consuming, and are ineffective for detecting drug-resistant bacteria in a low metabolic state. The development of biotechnology, especially the widespread application of fluorescent labeling technology, has provided new ideas for bacterial detection and monitoring.

[0004] Fluorescent proteins, especially green fluorescent protein (GFP), are widely used in the field of biomarkers and monitoring due to their advantages such as easy detection, non-toxicity and good stability. However, the traditional strong fluorescent protein labeling method has limitations in detecting bacteria in a low metabolic state, because even if the bacteria are in a dormant or low metabolic state, the strong fluorescent protein is retained due to its stability, resulting in an inability to accurately distinguish between actively metabolizing bacteria and low metabolic resistant bacteria. Therefore, there is an urgent need in this field for a method that can accurately detect low metabolic resistant bacteria to address the shortcomings of traditional bacterial detection kit monitoring methods in identifying low metabolic resistant bacteria and provide new technical means for monitoring and controlling bacterial resistance. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a non-stable GFP heavy-labeled Escherichia coli strain and its application.

[0006] To achieve the above objectives, the present invention provides an unstable GFP re-labeled Escherichia coli strain, comprising a fusion expression unit of the rrnBP1 promoter and the unstable green fluorescent protein gene, wherein the unstable green fluorescent protein gene is a mutant of the ASV degradation tag coding sequence connected to the C-terminus of the gfpmut3b gene.

[0007] Furthermore, the rrnBP1 promoter has a nucleotide sequence as shown in SEQ ID No.1.

[0008] Furthermore, the amino acid sequence of the ASV degradation tag is shown as SEQ ID No. 2.

[0009] Furthermore, the fusion expression unit is cloned into the pET28a vector backbone.

[0010] The present invention also provides a use of the above-mentioned unstable GFP heavily labeled Escherichia coli strain in detecting low-metabolism drug-resistant bacteria.

[0011] The present invention also provides a method for detecting low-metabolism drug-resistant bacteria, comprising the following steps:

[0012] (a) inoculating the unstable GFP-relabeled Escherichia coli strain according to any one of claims 1 to 4 into a bacterial sample to be tested;

[0013] (b) shaking culture;

[0014] (c) Detecting the fluorescence intensity. When the fluorescence intensity decays to less than 40% of the peak intensity, it indicates the presence of low-metabolism drug-resistant bacteria.

[0015] Furthermore, in step (b), antibiotics are added during the culture process, and the antibiotics are selected from tetracyclines, β-lactams or aminoglycosides, and the concentration is sub-inhibitory concentration.

[0016] Furthermore, the culture conditions are 37° C., 160 rpm, and 1-14 h.

[0017] The present invention also provides a recombinant vector for constructing the unstable GFP Escherichia coli heavy-labeled strain according to claim 1, comprising an operable connection between the rrnBP1 promoter and the gfp-asv gene, and the recombinant vector is pET28a-gfpasv.

[0018] The present invention also provides a kit comprising the above-mentioned unstable GFP Escherichia coli heavy-labeled strain or the above-mentioned recombinant vector, an antibiotic solution and a fluorescence detection buffer.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] In order to detect low-metabolism and drug-resistant bacteria in wild bacterial populations, the present invention uses Escherichia coli as the research object, utilizes the ribosomal promoter rrnbp1 related to bacterial growth metabolism (when the bacteria are actively growing, the metabolism is high, and the rrnbp1 promoter efficiently activates downstream gene transcription; when the bacteria are in a state of growth stagnation, the metabolism is low, and the rrnbp1 promoter is inefficient or even unable to activate downstream gene transcription), and transforms the strong fluorescence of GFP into unstable fluorescence, constructing an unstable GFP strain that can respond to bacterial growth and metabolic activity with the rrnbp1 promoter, and detects low-metabolism and drug-resistant bacteria in the bacterial population by the intensity of fluorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison of growth phenotypic characteristics of different strains; a. Detection of growth phenotypic characteristics of wild-type strain; b. Detection of growth phenotypic characteristics of unstable Prrnbp1-GFP heavily labeled strain; c. Detection of growth phenotypic characteristics of strong fluorescent GFP labeled strain;

[0022] Figure 2 Bacterial growth of the wild-type strain, the unstable Prrnbp1-GFP heavily labeled strain, and the strongly fluorescent GFP labeled strain was measured;

[0023] Figure 3 The fluorescence intensity changes of the unstable Prrnbp1-GFP heavy-labeled strain and the strong GFP-labeled strain at different growth times;

[0024] Figure 4 Fluorescence intensity detection of the unstable Prrnbp1-GFP re-labeled strain at different growth stages; a. Fluorescence intensity of the strain after 2 hours of growth; b. Fluorescence intensity of the strain after 4 hours of growth; c. Fluorescence intensity of the strain after 8 hours of growth; d. Fluorescence intensity of the strain after 12 hours of growth;

[0025] Figure 5 Comparison of fluorescence intensity of the unstable Prrnbp1-GFP heavily labeled strain before and after antibiotic treatment; a. Fluorescence intensity detection of the control group; b. Fluorescence intensity detection of the treated group;

[0026] Figure 6 Schematic diagram of the rrnBP1 promoter sequence structure;

[0027] Figure 7 Schematic diagram of the structure of the final vector pET28a-gfpasv. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] Experimental Materials:

[0030] Bacteria: E. coli DH5α was selected as the recipient bacterium, and pET28a vector, E. coli S17λpir and a strain containing the gfpmut3b plasmid were prepared as template sources.

[0031] Reagents: T4 DNA ligase, restriction endonucleases (SphI and NdeI), buffer solution required for PCR reaction, dNTPMix, primers (S1, S2, P1, P2, gfp-1, gfp-2), rTaq enzyme, DNA template extraction reagent, agarose gel electrophoresis reagent, DNA gel recovery kit, LB medium, etc.

[0032] Example 1 Construction of metabolically responsive vector pET28a-gfpasv

[0033] Step 1: Amplification and cloning of the rrnBP1 promoter

[0034] Using Escherichia coli genomic DNA as a template, the rrnBP1 promoter was amplified in two stages by overlap extension PCR. The overlapping long primers S1 / S2 were used as templates to amplify the fragment PS1. The PS1 fragment plasmid was used as a template and primers P1 / P2 were used to amplify the rrnBP-1 promoter fragment (M28522.1, the nucleotide sequence is shown in SEQ ID No. 1, containing the rRNArrnB core promoter sequence, UP element, Fis binding site and RBS, as shown in SEQ ID No. 1). Figure 6 shown).

[0035] SEQ ID No. 1:

[0036] AGGCTGATTTGGTTGAATGTTGCGCGGTCAGAAAATTATTTTAAATTTCCTCTTGTC AGGCCGGAATAACTCCCTATAATGCGCCACCA

[0037] The primer sequences are as follows:

[0038] S1: GCATGC AATTGTGAGCGGATAACAATTA TTGGTTGAATGTTGCGCGGTCAGAA AATTATTTTAA ATTTCCTCTTGTCAGGCCGGAATAACT (NdeI)

[0039] S2: CATATGTATATCTCCTTCTTAAAGTTAAACAAGGGAATTGTTATCCGCTCACAA TTGGTGGCGCATTATAGGGAGTTATTCCGGCCTGACAA(SphI)

[0040] P1: CATATG TATATCTCCTTCTT(SphⅠ)

[0041] P2: GCATGC ATTGGTTGAATGTTGCGC(NdeⅠ)

[0042] The PCR reaction system is as follows:

[0043]

[0044]

[0045] PCR amplification program: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min (30×); 72°C for 5 min; 16°C∞.

[0046] The products obtained by P1 / P2 amplification were electrophoresed on 2% agarose gel and recovered. The purified fragments were ligated into the pMD19-T vector and transformed into E. coli DH5α. The recombinant plasmid PrrnBP-1 was obtained by blue-white spot screening and sequencing verification.

[0047] Step 2: Expression vector assembly

[0048] PrrnBP-1 and the pET28a vector (Thermo Fisher, Catalog No. 69864-3) were double-digested with the restriction endonucleases SphI and NdeI and reacted at 37°C for 3 hours. The rrnBP1 fragment and the linearized pET28a vector were recovered from gel electrophoresis and ligated with T4 DNA ligase (Takara, Catalog No. 2011A) at 16°C for 12 hours. The ligation product was transformed into E. coli DH5α, and positive colonies were screened on LB plates containing kanamycin (50 μg / mL) to obtain the recombinant expression vector pET28a-rrnBP1.

[0049] Step 3: Unstable GFP gene insertion

[0050] Using the gfpmut3b plasmid (Addgene, Cat. No. 5457) as a template, primers containing the degradation tag ASV (SEQ ID No. 2) were designed:

[0051] gfp-1: 5'-ATATACATATGGTAAAGGAGAAGAACTTTTCA-3' (NdeI site)

[0052] gfp-2: 5'-CTCTCTGCTCGAGTATTAAACTGATGCAGCGTAG...-3' (XhoI site)

[0053] PCR amplification conditions were the same as in step 1 to obtain the gfp-asv fragment. After double digestion with NdeI / XhoI, it was ligated with the pET28a-rrnBP1 vector digested with the same enzymes to obtain the final vector pET28a-gfpasv (see the schematic diagram for the structure). Figure 7 ).

[0054] Degradation tag ASV SEQ ID No. 2:

[0055] AANDENYAASV.

[0056] The nucleotide sequence of gfpmut3b plasmid is shown in SEQ ID No. 3:

[0057] ttatttgtatagttcatccatgccatgtgtaatcccagcagctgttacaaactcaagaaggaccatgtggtctctcttttcgttgggatctttcgaaagggcagattgtgtggacaggtaatggttgtctggtaaaaggacagggccatcgccaattggagtattttgttgataatggtctgctagttgaacgcttccatcttcaatgttgtgtctaattttgaagttaactttgattccattcttttgtttgtctgccatgatgtatacattgtgtgagttatagttgtattccaatttgtgtccaagaatgtttccatcttctttaaaatcaataccttttaactcgattctattaacaagggtatcaccttcaaacttgacttcagcacgtgtcttgtagttcccgtcatctttgaaaaatatagttctttcctgtacataaccttcgggcatggcactcttgaaaaagtcatgccgtttcatatgatctgggtatctcgcaaagcattgaacaccataaccgaaagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtaagttttccgtatgttgcatcaccttcaccctctccactgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaa aagttcttctcctttacgcat。

[0058] The amino acid sequence expressed by the gfp - asv fragment is as shown in SEQ ID No.4:

[0059] MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKRPAANDENYAASV.

[0060] Example 2 Construction and phenotypic verification of engineered strains

[0061] Step 1: Strain transformation and culture

[0062] pET28a-gfpasv was electroporated into E. coli BL21(DE3), plated onto LB plates containing kanamycin (50 μg / mL), and cultured at 37°C for 16 hours. A single colony was selected and inoculated into 5 mL of LB liquid medium and cultured at 37°C, 200 rpm, and shaken for 12 hours to prepare the seed culture.

[0063] Step 2: Growth curve and fluorescence dynamic monitoring

[0064] Inoculate the seed solution into 100 mL of LB medium (containing kanamycin) at a dilution of 1:1000 and culture at 37°C and 160 rpm with shaking. Take samples every hour:

[0065] Colony count: Take 100 μL of sample and serially dilute it, then spread it on the plate and incubate it at 37°C for 24 hours to count the CFU;

[0066] OD600 determination: 200 μL of sample was added to a 96-well plate, and the absorbance was detected using a microplate reader (BioTek Synergy H1);

[0067] Fluorescence intensity detection: 1 mL of sample was taken simultaneously, centrifuged and resuspended in PBS, and relative fluorescence units (RFU) were measured using a fluorescence spectrophotometer (Ex = 488 nm, Em = 509 nm). The results were standardized as RFU / OD600.

[0068] result:

[0069] 1 Comparison of growth phenotypic characteristics of different strains

[0070] Phenotypic characteristics of active wild-type E. coli strain, unstable Prrnbp1-GFP heavy-labeled strain and strong fluorescent GFP-labeled strain are shown in Figure 1As shown, the wild-type strain was used as the control group ( Figure 1 a), the growth phenotype characteristics of the strain were compared with those of the unstable Prrnbp1-GFP heavy-labeled strain ( Figure 1 b) and strains with strong fluorescent GFP markers ( Figure 1 c) Growth phenotypic characteristics. Both the unstable Prrnbp1-GFP heavily labeled strain and the strongly fluorescent GFP labeled strain showed good growth.

[0071] 2 Changes in bacterial cell numbers at different growth stages of different strains

[0072] Measure the OD of different strains 1-14h 600 The change curve is as follows Figure 2 As shown in the figure, within the bacterial growth time of 1-7 hours, the bacterial number showed an increasing trend. The number of wild-type E. coli strains, unstable Prrnbp1-GFP heavily labeled strains, and strongly GFP labeled strains was almost the same, which did not affect practical use.

[0073] 3 Comparison of fluorescence intensity of strains at different culture stages

[0074] The fluorescence color development of the unstable Prrnbp1-GFP heavy-labeled strain and the strong GFP-labeled strain at different growth times is shown in Figure 2. Figure 3 As shown, during the 1-12 hours of E. coli culture, the fluorescence color of the strongly GFP-labeled strain remained stable, with almost no change. The fluorescence intensity of the unstable Prrnbp1-GFP heavily labeled strain gradually increased within 1-8 hours, stabilized between 8-11 hours, and gradually weakened after 11 hours, showing a downward trend, indicating the presence of low-metabolism bacteria in the colony.

[0075] Subsequently, the fluorescence of the unstable Prrnbp1-GFP heavy-labeled strain was detected at 2h, 4h, 8h and 12h of growth. Figure 4 As shown, after 2 h of growth, the bacteria were in a slow growth stage and the fluorescence intensity was weak ( Figure 4 a); At 4h and 8h of growth, the bacteria are in the logarithmic growth phase, the number increases rapidly, the growth is active, and the fluorescence intensity becomes stronger ( Figure 4 b, Figure 4 c); After 12 hours of growth, the bacterial growth is in a stable stage, and the bacterial colony begins to enter a low metabolic dormancy state, and the fluorescence intensity decreases ( Figure 4 d).

[0076] Example 3 Detection of low-metabolism drug-resistant bacteria under antibiotic treatment

[0077] The non-stable Prrnbp1-GFP heavily labeled Escherichia coli strains grown naturally for 4 hours were divided into two groups:

[0078] Control group: continued routine culture;

[0079] Treatment group: Tetracycline was added to a final concentration of 20 μg / mL and cultured at 37°C with shaking for 2 hours.

[0080] Samples were taken for fluorescence microscopy observation (Nikon Eclipse Ti2, GFP filter) and fluorescence intensity quantification.

[0081] Comparison of fluorescence intensity of the unstable Prrnbp1-GFP heavy-labeled strain before and after antibiotic treatment Figure 5 As shown, the control group was not added with tetracycline for bacterial culture, and the strain was in a metabolically active state, and its fluorescence intensity was detected to be strong ( Figure 5 a); After the treatment group was treated with tetracycline for bacterial culture, the active normal bacterial flora became resistant due to the induction of antibiotics and were in a low metabolic dormant state. Therefore, the fluorescence intensity of the strain was weakened after the addition of tetracycline ( Figure 5 b).

[0082] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A non-stable GFP heavy-labeled Escherichia coli strain, characterized in that: The invention comprises a fusion expression unit of rrnBP1 promoter and unstable green fluorescent protein gene, wherein the unstable green fluorescent protein gene is a mutant of ASV degradation tag coding sequence connected to the C-terminus of gfpmut3b gene.

2. The unstable GFP re-labeled Escherichia coli strain according to claim 1, characterized in that The rrnBP1 promoter has a nucleotide sequence as shown in SEQ ID No.

1.

3. The unstable GFP re-labeled Escherichia coli strain according to claim 1, characterized in that The amino acid sequence of the ASV degradation tag is shown in SEQ ID No.

2.

4. The unstable GFP re-labeled Escherichia coli strain according to claim 1, characterized in that The fusion expression unit was cloned into the pET28a vector backbone.

5. Use of the unstable GFP re-labeled Escherichia coli strain according to any one of claims 1 to 4 in the preparation of a reagent for detecting low-metabolism drug-resistant bacteria.

6. A method for detecting low-metabolism drug-resistant bacteria, characterized in that: The following steps are involved: (a) inoculating the unstable GFP-relabeled Escherichia coli strain according to any one of claims 1 to 4 into a bacterial sample to be tested; (b) shaking culture; (c) Detecting the fluorescence intensity. When the fluorescence intensity decays to less than 40% of the peak intensity, it indicates the presence of low-metabolism drug-resistant bacteria.

7. The method according to claim 6, characterized in that In step (b), antibiotics are added during the culture process, wherein the antibiotics are selected from tetracyclines, β-lactams or aminoglycosides, and the concentration is sub-inhibitory concentration.

8. The method according to claim 6, characterized in that The culture conditions are 37° C., 160 rpm, and 1-14 h.

9. A recombinant vector for constructing the unstable GFP-relabeled Escherichia coli strain according to claim 1, characterized in that: The rrnBP1 promoter and the gfp-asv gene are operably connected, and the recombinant vector is pET28a-gfpasv.

10. A kit, characterized in that The method comprises the unstable GFP re-labeled Escherichia coli strain according to any one of claims 1 to 4 or the recombinant vector according to claim 9, as well as an antibiotic solution and a fluorescence detection buffer.

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