Double-fluorescence-labeled engineering bacterium for in-situ tracing of horizontal transfer of plasmids in pseudomonas between bacteria as well as construction method and application of double-fluorescence-labeled engineering bacterium

Abstract

The invention discloses a double-fluorescence-labeled engineering bacterium for in-situ tracing of horizontal transfer of plasmids in pseudomonas among bacteria as well as a construction method and application of the double-fluorescence-labeled engineering bacterium, pseudomonas is taken as an original strain, a green fluorescence strong expression component is integrated into a pseudomonas chromosome genome, and green fluorescence-labeled pseudomonas is obtained; a red fluorescence strong expression element is inserted into a multiple cloning site of a broad-host zygote, and is converted into the green fluorescence labeled pseudomonas to form a double-fluorescence labeled engineering bacterium. The double-fluorescence-labeled strain disclosed by the invention can generate corresponding green fluorescence or red fluorescence after being excited by a green or red fluorescence excitation wavelength; when the plasmids enter other bacteria under the action of conjugation or transformation, the recipient bacteria can only present red fluorescence under the excitation of the red fluorescence excitation wavelength, and the horizontal transfer condition of the plasmids in the pseudomonas bacteria among the bacteria under the in-situ condition can be accurately tracked through the intuitive method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a double-fluorescent-labeled engineering bacterium used for in-situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas, and a construction method and application thereof. Background Art

[0002] Fluorescence tracing is a widely used detection method in biological and medical research. It uses fluorescent markers to track and observe the behavior of biological molecules or cells. In the field of bacteriology, fluorescence tracing can be used to study plasmid transfer between bacteria, which is an important mechanism for the exchange of genetic material and is of great significance for the adaptive evolution of bacteria and the spread of antibiotic resistance genes.

[0003] Fluorescent protein-based fluorescence tracing technology is to introduce fluorescent protein genes into bacteria. After the expressed fluorescent protein molecules absorb light energy of a specific wavelength, they will be excited to a higher energy state, and then release light of a specific wavelength in the process of returning to the ground state. By detecting this emitted light, the fluorescently labeled bacteria can be located and observed. By inserting the fluorescent gene into plasmid DNA, a fluorescently labeled plasmid can be created, so that the distribution and transfer of the plasmid in the bacterial population can be directly observed under a microscope; at the same time, by inserting the fluorescent gene into the chromosomal DNA, the plasmid donor strain can be stably marked, which is conducive to observing the real-time situation of the donor strain spreading the plasmid to the outside world. This is crucial to understanding how plasmids spread in bacterial populations. At present, the original host strain cannot be observed by fluorescence in the existing technology, and only the luminescence of the recipient strain after receiving the plasmid can be observed, and the fluorescence expression in the existing recipient bacteria is weak. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a dual-fluorescence-labeled engineered bacterium for in situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas. Under a fluorescence microscope, the dual-fluorescence-labeled strain can produce corresponding green or red fluorescence after being excited by a green or red fluorescence excitation wavelength; and when the plasmid enters other bacteria through conjugation or transformation, the recipient bacteria can only exhibit red fluorescence under the excitation of a red fluorescence excitation wavelength. Through this intuitive method, the horizontal transfer of plasmids between bacteria in Pseudomonas can be accurately tracked in situ. The present invention can simultaneously observe the fluorescence in the host bacteria and the recipient, effectively solving the problem in the prior art that the host cannot be observed by fluorescence, and the recipient bacteria are difficult to observe after receiving the plasmid due to weak fluorescence. Thereby, the horizontal transfer of genes in a visual environment can be achieved quickly and effectively.

[0005] The present invention also provides a double fluorescence labeling method for in-situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas; and an in-situ tracing method for the horizontal transfer of plasmids between bacteria in Pseudomonas.

[0006] Technical solution: To achieve the above-mentioned purpose, the present invention provides a dual-fluorescence-labeled engineered bacterium for in situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas. Pseudomonas is used as a starting strain, and the green fluorescence strong expression component of PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette is integrated into the chromosomal genome of Pseudomonas to obtain green fluorescence-labeled Pseudomonas; PA1 / 04 / 03-RBSII-RFP is inserted into the multiple cloning site of the broad-host conjugative plasmid pBBR1MCS-4, and it is transformed into the above-mentioned green fluorescence-labeled Pseudomonas to form a dual-fluorescence-labeled engineered bacterium.

[0007] Wherein, the Pseudomonas is a Pseudomonas syringae strain.

[0008] The green fluorescence strong expression component of the PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette includes the PA1 / 04 / 03 promoter, the RBSII ribosome binding site, the gfp gene, and the T0-T1 terminator, and its sequence is shown in SEQ ID NO.1.

[0009] The PA1 / 04 / 03-RBSII-RFP includes the PA1 / 04 / 03 promoter, the RBSII ribosome binding site, and the RFP gene, and its sequence is shown in SEQ ID NO.2.

[0010] Preferably, the dual-fluorescence-labeled engineered bacteria can produce corresponding green or red fluorescence after being excited by a green or red fluorescence excitation wavelength.

[0011] The method for constructing a dual-fluorescence-labeled engineered bacterium for in situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas comprises the following steps:

[0012] (1) Construction of green fluorescence labeled Pseudomonas strain: Synthesize the green fluorescence strong expression component PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette and insert it into the Not I restriction site of the pUT4K-mini-Tn5 plasmid; introduce the plasmid into Pseudomonas by diparental conjugation, and mini-Tn5 carrying PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette jumps and integrates into the Pseudomonas chromosome genome, forming a Pseudomonas single fluorescence labeled strain that stably expresses green fluorescence;

[0013] (2) Construction of a green-red dual fluorescence labeled Pseudomonas strain. The red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP was synthesized and inserted into the multiple cloning site of the broad host conjugative plasmid pBBR1MCS-4 by enzyme digestion. The plasmid was then introduced into the green fluorescent Pseudomonas single fluorescence labeled strain through transformation to form a green-red dual fluorescence labeled Pseudomonas strain.

[0014] In step (2), the red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP is synthesized and inserted into the multi-cloning sites Kpn I and EcoR I of the broad-host conjugative plasmid pBBR1MCS4 by enzyme digestion.

[0015] Preferably, the method for constructing a dual-fluorescence-labeled engineered bacterium for in situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas comprises the following steps:

[0016] (1) Construction of green fluorescence-labeled Pseudomonas strain: The green fluorescence strong expression component PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette was synthesized and inserted into the Not I restriction site of the pUT4K-mini-Tn5 plasmid. The plasmid was introduced into Pseudomonas by diparental conjugation, and the conjugates were screened on LB medium containing 20 mg / L kanamycin and 10 mg / L gentamicin. Mini-Tn5 carrying PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette jumped and integrated into the chromosome genome of Pseudomonas syringae, forming a single fluorescent-labeled Pseudomonas strain that stably expressed green fluorescence. Fluorescence was confirmed by fluorescence microscopy.

[0017] (2) Construction of green-red dual fluorescence labeled Pseudomonas strain: The red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP was synthesized and inserted into the multicloning site of the broad host conjugative plasmid pBBR1MCS-4 by enzyme digestion. The plasmid was then introduced into the green fluorescent single fluorescence labeled Pseudomonas strain through transformation, and the transformants were screened on LB medium containing 20 mg / L kanamycin, 10 mg / L gentamicin, and 100 mg / L ampicillin. The generated green-red dual fluorescence labeled Pseudomonas strain was confirmed by fluorescence microscopy.

[0018] Preferably, the LB medium comprises: 20-30 g agar, 10 g NaCl, 10 g peptone, 5 g yeast powder, 1000 mL deionized water, adjusted to pH 7.2-7.4, and sterilized at 120° C. for 20 min.

[0019] The invention discloses an application of the double-fluorescent-labeled engineering bacteria for in-situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas in order to realize the tracking of the horizontal transfer of plasmids between bacteria.

[0020] The application process is as follows: the engineered bacteria with dual fluorescence labels are used as donor bacteria. Through conjugation transfer experiments, the donor bacteria transfer the plasmid containing the red fluorescence gene to the recipient bacteria. The recipient bacteria produce red fluorescence but not green fluorescence, thereby realizing the tracking of the horizontal transfer of the plasmid between bacteria.

[0021] Among them, green-red dual fluorescence is used to mark the Pseudomonas host body; only red fluorescence without green fluorescence is used to indicate the recipient strain after horizontal transfer of plasmid.

[0022] Furthermore, an in situ tracing method for horizontal transfer of plasmids between bacteria in Pseudomonas is used. Through conjugation experiments, a plasmid carrying a red fluorescent protein gene in a green-red dual-fluorescence-labeled Pseudomonas strain is transferred to a recipient strain. The recipient strain produces red fluorescence but not green fluorescence, while the donor bacteria itself produces both red and green fluorescence, thereby achieving in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas.

[0023] As an alternative, the specific steps of the in situ tracing method are as follows: take 1×10 8 CFU / ml of donor double fluorescent labeled Pseudomonas and 1×10 8 CFU / ml of each recipient bacteria 1ml, mix well, centrifuge to remove the supernatant; add 2ml sterile water to resuspend the bacteria, mix well, centrifuge to remove the supernatant; add 100μl liquid LB medium to resuspend the bacteria. Take a 0.22μm sterile filter membrane and place the filter membrane on the solid culture medium of the recipient bacteria; load all the resuspended bacteria in the center of the filter membrane and culture at 30℃ for 1d; wash the bacteria on the filter membrane with 2mL sterile water into a centrifuge tube, centrifuge, retain 200μl of the bacterial solution, and apply 100μl each to the solid culture medium of the recipient bacteria containing 50mg / l chloramphenicol and 100mg / l ampicillin, culture at 30℃ for 2d, and then observe the fluorescence under a fluorescence microscope.

[0024] The present invention also provides a dual fluorescence labeling method for in situ tracing the horizontal transfer of plasmids between bacteria in Pseudomonas. The green fluorescence strong expression component carrying PA1 / 04 / 03-RBSII-GFP-T0-T1Cassette is integrated into the Pseudomonas chromosome genome by mini-Tn5 transposition to form a labeled strain expressing green fluorescence. Then, the red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP is inserted into the multi-cloning site of the wide host conjugation plasmid pBBR1MCS4, and the plasmid is introduced into the above-mentioned green fluorescence labeled Pseudomonas through transformation to form a dual strong fluorescence labeling strain. Under a fluorescence microscope, the dual-labeled strain can produce corresponding green or red fluorescence after being excited by a green or red fluorescence excitation wavelength; and when the plasmid enters other bacteria through conjugation or transformation, the recipient bacteria can only show red fluorescence under the excitation of the red fluorescence excitation wavelength. Through this intuitive method, the horizontal transfer of plasmids between bacteria in Pseudomonas can be accurately tracked in situ.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0026] The present invention constructs a Pseudomonas engineered bacterium with dual fluorescence labeling, and realizes green-red dual fluorescence labeling of Pseudomonas by means of conjugation transformation, and can distinguish donor strains (green-red fluorescence) and recipient strains (red fluorescence), and can trace the process of horizontal transfer of plasmids from Pseudomonas to other bacteria, and the recipient bacteria can only present red fluorescence under the excitation of red fluorescence excitation wavelength, and can accurately track the horizontal transfer of plasmids between bacteria in Pseudomonas in situ by intuitive methods. The present invention can intuitively observe the fluorescence in the host bacteria and the recipient at the same time, effectively solving the problem that the host bacteria cannot be observed by fluorescence, and the recipient bacteria are not easy to directly observe due to weak fluorescence after receiving the plasmid. Thereby, the horizontal transfer of genes in a visual environment is realized quickly and effectively.

[0027] The present invention constructs a specific dual-fluorescence-labeled engineered bacterium for in situ tracing of the horizontal transfer of plasmids between bacteria in Pseudomonas. The in situ tracing of the horizontal transfer of plasmids between bacteria can help to improve the efficiency of conjugation and transfer of drug-resistant plasmids, enhance sensitivity to bacteriophages, and deepen the mechanism of environmental pressure promoting the spread of antibiotic resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a fluorescence micrograph of the double-labeled strain Pseudomonas syringae;

[0029] Figure 2Fluorescence micrograph of the pBBR1MCS-4 recipient strain Pseudomonas putida KT2440 propagated by the double-marked strain. DETAILED DESCRIPTION

[0030] In order to further illustrate the present invention, the present invention is further described below in conjunction with embodiments and drawings.

[0031] Unless otherwise specified, the materials and reagents in the present invention are conventional materials and reagents available on the market.

[0032] The strains, plasmids, etc. used in the present invention are all known materials.

[0033] Among them, Pseudomonas syringae is Pseudomonas syringae DC3000 (Cuppels DA.1986.Generation and Characterization of Tn5 Insertion Mutations in Pseudomonas syringae pv.tomato.Appl Environ Microbiol 51:323-7.), (Hrppilus: a novel hrp-dependent bacterial surface appendage produced by Pseudomonas syringae DC3000. Proc Nat Acad Sci USA 94:3459-3464).

[0034] Pseudomonas putida KT2440 (Nelson KE. 2002. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440 Environ Microbiol. 4: 799-808.) All of the above were provided by Nanjing Agricultural University.

[0035] LB medium: NaCl 10g, peptone 10g, yeast powder 5g, deionized water 1000mL, adjust pH to 7.2-7.4, autoclave at 120℃ for 20min. Add 20-30g agar to solid LB.

[0036] pUT4K-mini-Tn5 plasmid (suicide plasmid pUT4K containing mini-Tn5, pUT / mini-Tn5): see reference (Wu Jun, Xu Jingliang, Hong Qing, Li Shunpeng, Construction of genetically stable genetically engineered bacteria for degrading 666 and carbendazim, Acta Microbiologica Sinica, 2008, 48(1): 45-50.).

[0037] pBBR1MCS4, Brand: LMAI Bio, Product Number: LM-8176.

[0038] Example 1

[0039] Fluorescence Labeling of Double-labeled Pseudomonas syringae

[0040] (1) Construction of green fluorescence labeled Pseudomonas strain: A green fluorescence strong expression component PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette was synthesized, and its sequence is shown in SEQ ID NO.1. It was inserted into the Not I restriction site of the pUT4K-mini-Tn5 plasmid. The plasmid was introduced into Pseudomonas syringae by diparental conjugation, and the conjugates were screened on LB medium containing 20 mg / L kanamycin and 10 mg / L gentamicin. The mini-Tn5 carrying PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette in the conjugates was screened and integrated into the chromosome genome of Pseudomonas syringae, forming a single fluorescent labeled strain of Pseudomonas that stably expresses green fluorescence, and the fluorescence was confirmed by observation under a fluorescence microscope.

[0041] (2) Construction of green-red dual fluorescence labeled Pseudomonas strain: Synthesize the red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP, whose sequence is shown in SEQ ID NO.2, and insert it into the multi-cloning site Kpn I and EcoR I of the broad host conjugative plasmid pBBR1MCS-4 by enzyme digestion. Then, the plasmid is introduced into the green fluorescent Pseudomonas single fluorescence labeled strain by transformation, and the transformants are screened on LB medium containing 20 mg / L kanamycin, 10 mg / L gentamicin, and 100 mg / L ampicillin. The correct transformants are screened, and the green-red dual fluorescence labeled Pseudomonas strain produced is confirmed by fluorescence microscopy observation ( Figure 1 ).

[0042] Example 2

[0043] Horizontal transfer of red fluorescent plasmid from double-labeled strain into Pseudomonas KT2440

[0044] Materials: Double-labeled strain and Pseudomonas KT2440. The double fluorescent labeled strain and Pseudomonas KT2440 prepared in Example 1 were streaked on LB solid culture medium containing 100 mg / l ampicillin and 50 mg / l chloramphenicol, respectively, and cultured at 30°C for 3 days; single colonies on the plate were transferred to LB liquid culture medium and cultured at 30°C and 250 rpm overnight. Fresh bacterial liquid was centrifuged at 10,000 rpm for 2 minutes at room temperature, the cells were collected, and the cells were washed twice with sterile water, and the culture medium was removed. 1×10 8 CFU / ml of the donor double fluorescent labeled bacterial solution and 1×10 8 Mix 1 ml of each of the KT2440 bacterial liquid of the recipient bacteria with CFU / ml, and remove the supernatant by centrifugation; resuspend the bacteria in 2 ml of sterile water, mix well, and remove the supernatant by centrifugation; resuspend the bacteria in 100 μl of liquid LB medium. Take a 0.22 μm sterile filter membrane and place it on the solid LB medium of the recipient bacteria; load all the resuspended bacteria in the center of the filter membrane and culture it statically at 30°C for 1 day; wash the bacteria on the filter membrane with 2 mL of sterile water into a centrifuge tube, centrifuge, and retain 200 μl of the bacterial liquid, and apply 100 μl each to the solid LB medium of the recipient bacteria containing 50 mg / l chloramphenicol and 100 mg / l ampicillin, and culture at 30°C for 2 days. Then observe the fluorescence under a fluorescence microscope to obtain a recipient strain marked with only red fluorescence. Figure 2 . From the above experiments, it can be seen that the present invention realizes green-red dual fluorescence labeling of Pseudomonas, and can distinguish donor strains (green-red fluorescence) and recipient strains (red fluorescence). The recipient bacteria can only show red fluorescence under the excitation of the red fluorescence excitation wavelength, and the horizontal transfer of plasmids between bacteria in Pseudomonas in situ can be accurately tracked by an intuitive method. The present invention can intuitively observe the fluorescence in the host bacteria and the recipient at the same time, effectively solving the problem that the host bacteria cannot be observed by fluorescence, and the recipient bacteria are not easy to directly observe after receiving the plasmid due to the weak fluorescence expressed.

Claims

1. A dual-fluorescence-labeled engineered bacterium for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas, characterized in that: Taking Pseudomonas as the starting strain, the PA1 / 04 / 03-RBSII-GFP-T0-T1Cassette green fluorescence strong expression component was integrated into the Pseudomonas chromosomal genome to obtain green fluorescence labeled Pseudomonas; the PA1 / 04 / 03-RBSII-RFP red fluorescence strong expression element was inserted into the multiple cloning site of the wide host conjugative plasmid pBBR1MCS-4, and it was transformed into the above green fluorescence labeled Pseudomonas to form a double fluorescence labeled engineered bacterium.

2. The dual-fluorescence-labeled engineered bacteria for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas according to claim 1, characterized in that: The Pseudomonas is a Pseudomonas syringae strain.

3. The dual-fluorescence-labeled engineered bacteria for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas according to claim 1, characterized in that: The green fluorescence strong expression component of the PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette includes a PA1 / 04 / 03 promoter, an RBSII ribosome binding site, a gfp gene, and a T0-T1 terminator, and its sequence is shown in SEQ ID NO.

1.

4. The dual-fluorescence-labeled engineered bacteria for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas according to claim 1, characterized in that: The PA1 / 04 / 03-RBSII-RFP includes a PA1 / 04 / 03 promoter, an RBSII ribosome binding site, and an RFP gene, and its sequence is shown in SEQ ID NO.

2.

5. The dual-fluorescence-labeled engineered bacteria for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas according to claim 1, characterized in that: The dual-fluorescence-labeled engineered bacteria can generate corresponding green or red fluorescence after being excited by a green or red fluorescence excitation wavelength.

6. A method for constructing a dual-fluorescence-labeled engineered bacterium for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas as claimed in claim 1, characterized in that: The steps include: (1) Construction of green fluorescence labeled Pseudomonas strain: Synthesize the green fluorescence strong expression component PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette and insert it into the NotI restriction site of the pUT4K-mini-Tn5 plasmid; introduce the plasmid into Pseudomonas by diparental conjugation, and mini-Tn5 carrying PA1 / 04 / 03-RBSII-GFP-T0-T1 Cassette jumps and integrates into the Pseudomonas chromosome genome, forming a Pseudomonas single fluorescence labeled strain that stably expresses green fluorescence; (2) Construction of a green-red dual fluorescence-labeled Pseudomonas strain: The red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP was synthesized and inserted into the multi-cloning sites Kpn I and EcoR I of the broad-host conjugative plasmid pBBR1MCS-4 by enzyme digestion. The plasmid was then introduced into the green fluorescent Pseudomonas single fluorescence-labeled strain through transformation to form a green-red dual fluorescence-labeled Pseudomonas strain.

7. Use of the dual-fluorescence-labeled engineered bacteria for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas as claimed in claim 1 in tracking horizontal transfer of plasmids between bacteria.

8. The use according to claim 7, characterized in that The application process is: using the double-fluorescent-labeled engineered bacteria of claim 1 as donor bacteria, and through a conjugation transfer experiment, the donor bacteria transfers the plasmid containing the red fluorescent gene to the recipient bacteria, and the recipient bacteria produces red fluorescence but no green fluorescence, thereby realizing the tracking of the horizontal transfer of the plasmid between bacteria.

9. The use according to claim 7, characterized in that: The recipient bacteria is preferably Pseudomonas putida KT2440.

10. A dual fluorescence labeling method for in situ tracing of horizontal transfer of plasmids between bacteria in Pseudomonas, characterized in that: The method comprises the following steps: integrating a green fluorescence strong expression component carrying PA1 / 04 / 03-RBSII-GFP-T0-T1Cassette into the chromosome genome of Pseudomonas by transposition to form a labeled strain expressing green fluorescence, and then inserting a red fluorescence strong expression element PA1 / 04 / 03-RBSII-RFP into the multiple cloning site of a broad-host conjugative plasmid pBBR1MCS-4, and introducing the plasmid into the green fluorescence-labeled Pseudomonas by transformation to form a double-strong fluorescence labeled strain, which can produce corresponding green or red fluorescence after being excited by a green or red fluorescence excitation wavelength; and when the plasmid enters other bacteria through conjugation or transformation, the recipient bacteria can only present red fluorescence under the excitation of a red fluorescence excitation wavelength, and this intuitive method can accurately track the horizontal transfer of plasmids between bacteria in Pseudomonas in situ.

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