Conjugative transfer donor bacteria and construction method and application thereof

Abstract

The present application relates to the technical field of conjugative transfer engineering bacteria, and relates to a conjugative transfer donor bacterium, a construction method and application thereof, the conjugative transfer donor bacterium is Escherichia fergusonii EFmg-sy, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M2025310;Or, the conjugative transfer donor bacterium is Escherichia fergusonii EFmg-cj, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M20251730.The conjugative transfer donor bacterium provided by the present application is stable in property, simple in operation as a conjugative transfer donor, can be quickly detected and distinguished from the receptor bacterium that has received plasmid through the method of fluorescence detection, and the detection of the plasmid horizontal transfer ability of Escherichia fergusonii is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of conjugative transfer engineering bacteria, and particularly relates to a conjugative transfer donor bacterium and a construction method and application thereof. BACKGROUND

[0002] Escherichia fergusonii is a member of the Enterobacteriaceae family and is a zoonotic opportunistic pathogen, which is widely distributed in the environment, food, human and animal intestines. Similar to other species of the Enterobacteriaceae family, E. fergusonii also has multiple drug resistance. In recent years, more and more literatures report the isolation of multiple drug-resistant E. fergusonii from animals and clinical cases, which contains high-risk resistant genes of some important antibiotics, such as mcr-1 , bla NDM and tet (X4), which brings great challenges to clinical treatment. E. fergusonii has strong ability to accept exogenous plasmids, and has low adaptive cost, which has the risk of spreading important resistant genes, so it is necessary to establish a high-efficiency and reliable method for detecting the transmission ability of resistant plasmids of E. fergusonii.

[0003] At present, the evaluation of the transmission efficiency of resistant genes is mainly through conjugation transfer experiment, and the commonly used receptor bacteria are E. coli J53 and C600. This traditional detection method cannot simulate the conjugation transfer environment under natural conditions, and cannot realize high-throughput detection. Therefore, it is urgent to develop a high-efficiency and sensitive detection method for plasmid transmission ability, to realize reliable evaluation of the plasmid transmission ability of E. fergusonii under natural conditions, and to provide data reference for the control of resistant gene transmission. SUMMARY

[0004] Therefore, in order to solve the above problems, the present application provides a conjugative transfer donor bacterium and a construction method and application thereof.

[0005] Therefore, in order to solve the above problems, the present application provides a conjugative transfer donor bacterium and a construction method and application thereof.

[0006] Or, the conjugative transfer donor bacterium is Escherichia fergusonii EFmg-cj, which has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:M20251730.

[0007] In another aspect, the present application provides a method for constructing a conjugative transfer donor bacterium, comprising the following steps: transferring a target plasmid into a target strain, wherein the method for constructing the target strain comprises the following steps of:

[0008] eliminating the endogenous plasmid pEFER of Escherichia fergusonii to obtain a first intermediate strain;

[0009] constructing bla TEM-1B a knockout strain, and the specific steps comprise the following: transferring the plasmid pEcCas into the competent cells of the first intermediate strain to form a second intermediate strain, transferring the plasmid pTAKB into the competent cells of the second intermediate strain to form a third intermediate strain, eliminating the plasmid pTAKB in the third intermediate strain to form a fourth intermediate strain, and the gene sequence of the plasmid pTAKB is shown in SEQ ID NO: 2;

[0010] transferring the plasmid pTAMK into the fourth intermediate strain, eliminating the plasmid pTAMK and the plasmid pEcCas to form the target strain, and the gene sequence of the plasmid pTAMK is shown in SEQ ID NO: 3;

[0011] The gene sequence of the target plasmid consists of SEQ ID NO: 4, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63 in the 5'-3' direction or consists of SEQ ID NO: 45, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70 in the 5'-3' direction.

[0012] In some embodiments, the sequence of the primer for verifying the loss of the endogenous plasmid in the first intermediate strain is shown in SEQ ID NO: 5-SEQ ID NO: 6.

[0013] In some embodiments, the sequence of the primer for verifying the knockout of the gene in the fourth intermediate strain is shown in SEQ ID NO: 7-SEQ ID NO: 8. bla TEM-1B In some embodiments, the sequence of the primer for screening the fourth intermediate strain containing the plasmid pTAMK is shown in SEQ ID NO: 9-SEQ ID NO: 10.

[0014] In some embodiments, the sequence of the primer for verifying the elimination of the plasmid pTAMK in the third intermediate strain and / or the fourth intermediate strain is shown in SEQ ID NO: 11-SEQ ID NO: 12.

[0015] In some embodiments, the sequence of the primer for verifying the elimination of the plasmid pTAMK in the third intermediate strain and / or the fourth intermediate strain is shown in SEQ ID NO: 11-SEQ ID NO: 12.

[0016] In some embodiments, the primer sequences for verifying that the fourth intermediate strain eliminates plasmid pEcCas are shown in SEQ ID NO: 13-SEQ ID NO: 14.

[0017] In some embodiments, the method for constructing the target plasmid includes the following steps: using E. coli ECCRA-119 as a template, extracting plasmid pTB203, transforming the extracted plasmid pTB203 into E. coli DH10B, culturing and screening to obtain a strain containing plasmid pTB203, extracting the plasmid to obtain plasmid pTB203, and the gene sequence of the plasmid pTB203 consists of SEQ ID NO: 15, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66 in the 5'-3' direction;

[0018] The plasmid pEcCas and the plasmid pTAG are transformed into E. coli DH10B competent cells, and the fifth intermediate strain is screened, and the gene sequence of the plasmid pTAG is shown in SEQ ID NO: 42;

[0019] The plasmid pTB203 is transformed into the competent cells of the fifth intermediate strain, and the plasmid pEcCas and the plasmid pTAG are eliminated by screening to obtain a strain containing the target plasmid, and the target plasmid is extracted.

[0020] In some embodiments, the method for constructing the target plasmid includes the following steps: using E. coli EF44 as a template, extracting plasmid pTB44P3, transforming the extracted plasmid pTB44P3 into E. coli DH10B, culturing and screening to obtain a strain containing plasmid pTB44P3, extracting the plasmid to obtain plasmid pTB44P3, and the gene sequence of the plasmid pTB44P3 consists of SEQ ID NO: 48, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75 in the 5'-3' direction;

[0021] The pEcCas and the pTAGmcr are transformed into E. coli DH10B competent cells, and the sixth intermediate strain is screened, and the sequence of the plasmid pTAGmcr is shown in SEQ ID NO: 47;

[0022] The plasmid pTB44P3 is transformed into the competent cells of the sixth intermediate strain, and the plasmid pEcCas and the plasmid pTAGmcr are eliminated by screening to obtain a strain containing the target plasmid, and the target plasmid is extracted.

[0023] In some embodiments, the primer sequence for verifying the plasmid pTB203 contained in the strain containing the plasmid pTB203 is shown in SEQ ID NO: 16-SEQ ID NO: 17.

[0024] In some embodiments, the primer sequence for verifying the plasmid pEcCas introduced into the fifth intermediate strain is shown in SEQ ID NO: 13-SEQ ID NO: 14.

[0025] In some embodiments, the primer sequence for verifying the plasmid pTB203 introduced into the fifth intermediate strain is shown in SEQ ID NO: 18-SEQ ID NO: 19.

[0026] In some embodiments, the primer sequence for verifying the plasmid pEcCas eliminated from the strain containing the target plasmid is shown in SEQ ID NO: 13-SEQ ID NO: 14.

[0027] In some embodiments, the primer sequence for verifying the plasmid pTAG or plasmid pTB44P3 eliminated from the strain containing the target plasmid is shown in SEQ ID NO: 11-SEQ ID NO: 12.

[0028] In some embodiments, the primer sequence for verifying the plasmid pTB44P3 contained in the sixth intermediate strain is shown in SEQ ID NO: 18-SEQ ID NO: 19.

[0029] In some embodiments, the method for constructing the plasmid pTAKB comprises the following steps: using the primer sequence shown in SEQ ID NO: 20-SEQ ID NO: 21 to amplify the first upstream homologous arm, and using the primer sequence shown in SEQ ID NO: 22-SEQ ID NO: 23 to amplify the first downstream homologous arm, using the plasmid pTA as a template;

[0030] using the primer sequence shown in SEQ ID NO: 24-SEQ ID NO: 25 to amplify the target plasmid backbone fragment, and using the primer sequence shown in SEQ ID NO: 26-SEQ ID NO: 27 to amplify the second plasmid backbone fragment, using the plasmid pTA as a template;

[0031] connecting the first upstream homologous arm, the first downstream homologous arm, the target plasmid backbone fragment, and the second plasmid backbone fragment, and screening to obtain the plasmid pTAKB.

[0032] In some embodiments, the method for constructing the plasmid pTAMK includes the following steps: using Escherichia coli Fergusonii as a template, amplifying the second upstream homologous arm using primers with sequences as shown in SEQ ID NO: 28~SEQ ID NO: 29, and amplifying the second downstream homologous arm using primers with sequences as shown in SEQ ID NO: 30~SEQ ID NO: 31;

[0033] With plasmid pET-28a(+)- mCherry Using primers with sequences as shown in SEQ ID NO: 32~SEQ ID NO: 33 as templates, amplification was performed. lacI q -pLpp-mCherry-Km R Integration fragment, plasmid pET-28a(+)- mCherry The sequence consists of SEQ ID NO: 46 and SEQ ID NO: 71 in the 5'-3' direction;

[0034] Using pTA plasmid as a template, the third plasmid backbone fragment was amplified using primers with sequences as shown in SEQ ID NO: 24 and SEQ ID NO: 35, and the fourth plasmid backbone fragment was amplified using primers with sequences as shown in SEQ ID NO: 34 and SEQ ID NO: 26. The gene sequence of the plasmid pTA is shown in SEQ ID NO: 44.

[0035] Using the third and fourth plasmid backbone fragments as templates, and primers with sequences as shown in SEQ ID NO: 24 and SEQ ID NO: 26, the third and fourth plasmid backbone fragments were fused by fusion PCR to obtain the fusion fragment.

[0036] The second upstream homologous arm, the second upstream homologous arm, lacI q -pLpp-mCherry-Km R The integrated fragment and the first fusion fragment were ligated and screened to obtain plasmid pTAMK, the sequence of which is shown in SEQ ID NO: 3.

[0037] In some embodiments, the method for constructing the plasmid pTAG includes the following steps: using plasmid pTB203 as a template, amplifying the third upstream homologous arm using primers with sequences as shown in SEQ ID NO: 36~SEQ ID NO: 37, and amplifying the third downstream homologous arm using primers with sequences as shown in SEQ ID NO: 38~SEQ ID NO: 39. The sequence of plasmid pTB203 is composed of SEQ ID NO: 15, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66 in the 5'-3' direction.

[0038] With plasmid pET-28a(+)- gfp Using primers with sequences as shown in SEQ ID NO: 40~SEQ ID NO: 41 as templates, amplification was performed. gfp The integrated fragment, the plasmid pET-28a(+)- gfp The sequence is shown in SEQ ID NO: 43;

[0039] Using pTA plasmid as a template, the third plasmid backbone fragment was amplified using primers with sequences as shown in SEQ ID NO: 32~SEQ ID NO: 33, and the fourth plasmid backbone fragment was amplified using primers with sequences as shown in SEQ ID NO: 34~SEQ ID NO: 35. The gene sequence of the pTA plasmid is shown in SEQ ID NO: 44.

[0040] Using the third and fourth plasmid backbone fragments as templates, and primers with sequences as shown in SEQ ID NO: 24 and SEQ ID NO: 26, the third and fourth plasmid backbone fragments were fused by fusion PCR to obtain the fusion fragment.

[0041] The third upstream homologous arm, the third downstream homologous arm, gfp The integrated fragment and the fused fragment were connected, screened, and the plasmid pTAG was obtained.

[0042] In some embodiments, the method for constructing plasmid pTAGmcr includes the following steps: using plasmid pTB44P3 as a template, amplifying the fourth upstream homologous arm using primers with sequences as shown in SEQ ID NO: 49~SEQ ID NO: 50, and amplifying the fourth downstream homologous arm using primers with sequences as shown in SEQ ID NO: 51~SEQ ID NO: 52;

[0043] With plasmid pET-28a(+)- gfp Using primers with sequences as shown in SEQ ID NO: 40~SEQ ID NO: 41 as templates, amplification was performed. gfpThe integrated fragment is obtained by connecting the fourth upstream homologous arm, the fourth downstream homologous arm, the third plasmid backbone fragment, the second plasmid backbone fragment, the first plasmid backbone fragment and the fusion fragment. gfp The sequence of the plasmid pET-28a(+) is shown as SEQ ID NO:43.

[0044] The target plasmid backbone fragment is amplified by using the pTA plasmid as a template and using primers with sequences shown as SEQ ID NO:24 and SEQ ID NO:54, and the fifth plasmid backbone fragment is amplified by using primers with sequences shown as SEQ ID NO:26 and SEQ ID NO:53.

[0045] The target plasmid backbone fragment and the fifth plasmid backbone fragment are fused by fusion PCR by using the target plasmid backbone fragment and the fifth plasmid backbone fragment as templates and using primers with sequences shown as SEQ ID NO:24 and SEQ ID NO:26.

[0046] The fourth upstream homologous arm, the fourth downstream homologous arm, gfp The integrated fragment is obtained by connecting the fourth upstream homologous arm, the fourth downstream homologous arm,

[0047] Meanwhile, the conjugative donor bacteria provided by the present application or the conjugative donor bacteria obtained by the method for constructing conjugative donor bacteria have the following uses:

[0048] (I) detecting the conjugative transfer ability of the donor bacteria and the recipient bacteria;

[0049] (II) distinguishing the conjugative donor bacteria and the recipient bacteria;

[0050] (III) detecting the transmission ability of the drug resistance gene;

[0051] (IV) evaluating the diversity of the recipient bacteria.

[0052] The technical scheme of the present application has the following advantages:

[0053] 1. The conjugative donor bacteria provided by the present application are Escherichia fergusonii EFmg-sy, which has been preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO:M2025310, or Escherichia fergusonii EFmg-cj, which has been preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO:M20251730. The conjugative donor bacteria provided by the present application have stable strain properties, are simple to operate as conjugative donors, can be quickly detected and distinguished from the recipient bacteria receiving plasmids by a fluorescence detection method, and thus the detection of the plasmid horizontal transfer ability of Escherichia fergusonii can be realized.

[0054] 2. The present invention provides a method for constructing a conjugation transfer donor bacterium, comprising the following steps: transferring a target plasmid into a target strain, wherein the method for constructing the target strain comprises the following steps: eliminating the endogenous plasmid pEFER of *Escherichia coli* to obtain a first intermediate strain; constructing... bla TEM-1B The knockout strain involves the following steps: first, plasmid pEcCas is transferred into competent cells of a first intermediate strain to form a second intermediate strain; second, plasmid pTAKB is transferred into competent cells of the second intermediate strain to form a third intermediate strain; third, plasmid pTAKB is eliminated from the third intermediate strain to form a fourth intermediate strain, the gene sequence of which is shown in SEQ ID NO: 2; fourth, plasmid pTAMK is transferred into the fourth intermediate strain, and plasmids pTAMK and pEcCas are eliminated to form the target strain, the gene sequence of which is shown in SEQ ID NO: 3; the gene sequence of the target plasmid is composed of SEQ ID NO: 4, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63 sequentially along the 5'-3' direction. This invention designs a gene sequence commonly expressed in the donor *Escherichia coli* Fergusoniae. mCherry and lacI Repressor protein genes, and the genes carrying the repressor proteins that regulate [the protein's function]. gfp The labeled plasmids under investigation can then be used to distinguish between red donor bacteria and green receiver bacteria by observing fluorescence. gfp This invention utilizes labeled recipient bacteria to study the plasmid-transmitting ability of *Escherichia coli*. The method is sensitive and reliable, and can simulate plasmid transmission under natural conditions. This invention provides technical support for the detection and control of drug resistance gene transmission and has high application value.

[0055] Escherichia fergusonii EFmg-sy has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025310, located at Wuhan University, Wuhan, China, 430072, China, on February 27, 2025.

[0056] Escherichia fergusonii EFmg-cj has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M20251730, located at Wuhan University, Wuhan, China, 430072, China, on July 30, 2025. Attached Figure Description

[0057] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings described below are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0058] Figure 1 is a schematic diagram of the plasmid pTAKB in the embodiment 1 of the present application;

[0059] Figure 2 is a schematic diagram of the plasmid pTAMK in the embodiment 1 of the present application;

[0060] Figure 3 is a schematic diagram of the plasmid pTAG in the embodiment 1 of the present application;

[0061] Figure 4 is a schematic diagram of the target plasmid in the embodiment 1 of the present application;

[0062] Figure 5 is the fluorescence microscope observation result of the target strain in the embodiment 1 of the present application, the scale is 20 μm;

[0063] Figure 6 is the fluorescence microscope observation result of the E. coli DH10B containing the target plasmid in the embodiment 1 of the present application, the scale is 20 μm;

[0064] Figure 7 is the fluorescence microscope observation result of the conjugation transfer donor bacteria in the embodiment 1 of the present application, the scale is 20 μm;

[0065] Figure 8 is the map of the pTAGmcr plasmid in the embodiment 2 of the present application;

[0066] Figure 9 is the map of the pTB44P3:: in the embodiment 2 of the present application; gfp plasmid;

[0067] Figure 10 is the fluorescence microscope observation result of 3.3 in the embodiment 2 of the present application;

[0068] Figure 11 is the fluorescence microscope observation result of 4.2 in the embodiment 2 of the present application;

[0069] Figure 12 is the fluorescence microscope observation result of the conjugation transfer donor bacteria transferred to the recipient bacteria in the embodiment of the present application, Figure 12 A strain DH10B-pTB203-gfp expressing green fluorescent protein in the embodiment of the present application, Figure 12B in the table indicates the strain EFmg-sy expressing red fluorescent protein, Figure 12 C in the table indicates the conjugation result of the strain EFmg-sy and the non-fluorescent recipient bacteria Escherichia coli C600, Figure 12 D in the table indicates the conjugation result of the strain EFmg-sy and the non-fluorescent Escherichia coli J53, Escherichia E in the table indicates the conjugation result of the strain EFmg and chicken fecal flora on the cellulose filter membrane. DETAILED DESCRIPTION

[0070] The following examples are provided to better further understand the present application and are not limited to the best mode contemplated, do not limit the scope of the application, and are not intended to convey any limitation as to subject matter and scope of the present application. Any person skilled in the art under the guidance of the present application or the combination of the present application with other prior art features can obtain any product identical or similar to the present application, which falls within the scope of protection of the present application.

[0071] The specific experimental steps or conditions not mentioned in the examples can be carried out according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not marked with the manufacturer, which are conventional reagent products that can be obtained by market purchase.

[0072] The standard strain of Escherichia fergusonii in the embodiments of the present application is Escherichia fergusonii (E. fergusonii). fergusonii Escherichia fergusonii The accession number is ATCC 35469.

[0073] The whole genome sequence of Escherichia coli ECCRA-119 can be seen from GenBank accession number CP029242~CP029245 (among them, CP029242 is the whole genome information of Escherichia coli ECCRA-119, and CP029243- CP029245 is the information of three plasmids contained in Escherichia coli ECCRA-119).

[0074] Escherichia coli DH10B is purchased from Shanghai Sangon Biological Engineering Co., Ltd.

[0075] The strain EF44 is disclosed from the literature Tang B, Chang J, Chen Y, Lin J, Xiao X, Xia X, Lin J, Yang H, Zhao G. E. fergusonii , an underrated repository for antimicrobial resistance in food animals. Microbiology Spectrum. 2022, 10(1):e0161721.

[0076] The primer information used in this embodiment is shown in Table 1, the strain information is shown in Table 2, the plasmid information is shown in Table 3, the PCR reaction system is shown in Table 4, and the PCR reaction procedure is shown in Table 5.

[0077] Table 1 Primer Information

[0078]

[0079] In Table 1, homologous arms are marked in italics and underlined, while gRNA is marked in bold underline.

[0080] Table 2. Strain Information

[0081]

[0082] Table 3 Plasmid Information

[0083]

[0084] Table 4 PCR reaction system

[0085]

[0086] Table 5 PCR reaction procedure

[0087]

[0088] Example 1

[0089] 1. Construction of edit plasmids

[0090] 1.1 Construction of pTAKB plasmid:

[0091] Escherichia coli of Ferguson ( bla Using the ATCC 35469 genome as a template, amplification was performed using primers bla-up-F (the homologous arm sequences on which the primers contain the bold underlined portion in SEQ ID NO.20 of Table 1), bla-up-R, and bla-down-F (the homologous arm sequences on which the primers contain the bold underlined portion in SEQ ID NO.22 of Table 1), and bla-down-R, respectively. Figure 1 TEM-1B The upstream and downstream homologous arms HA1 and HA2;

[0092] Using pTA plasmid (SEQ ID NO: 44) as a template, the plasmid backbone fragment Vbla-1 was amplified using primers V1-F and bla20-R (the gRNA sequence contained on the primers is the part in bold underlined in SEQ ID NO: 25 in Table 1), and the plasmid backbone fragment Vbla-2 was amplified using primer bla20-F (the gRNA sequence contained on the primers is the part in bold underlined in SEQ ID NO: 27 in Table 1) and V1-R.

[0093] The PCR reaction system described above is shown in Table 4, and the PCR reaction procedure is shown in Table 5.

[0094] The four fragments obtained above were recovered and their concentrations determined. They were then ligated using Gibson assembly, and the ligation products were transformed into *Escherichia coli* DH10B competent cells. The cells were incubated on LB agar plates containing 50 μg / mL apramycin sulfate (Apra) at 37°C for 15 h. Subsequently, plasmid extraction, enzyme digestion verification, and sequencing were performed using a kit to obtain the correctly constructed recombinant plasmid pTAKB. A schematic diagram of the recombinant plasmid pTAKB is shown below. E. fergusonii The sequence of plasmid pTAKB is SEQ ID NO:2.

[0095] 1.2 Construction of pTAMK plasmid:

[0096] Escherichia coli of Ferguson ( mCherry Using ATCC 35469 as a template, primers INg-up-F / R and INg-down-F / R were used to amplify the upstream and downstream homologous arms HA3 and HA4 of the integration site, respectively.

[0097] pET-28a(+)- mCherr Using mCherry-F / R primers as templates, amplification was performed. lacI y integrated fragment MK ( -pLpp-mCherry-Km q Figure 2 R );

[0098] Using pTA plasmid as a template, plasmid backbone fragment Vm-1 was amplified using primers V1-F and INg20-R, and plasmid backbone fragment Vm-2 was amplified using primers INg20-F and V1-R; using Vm-1 and Vm-2 as templates, plasmid backbone fragments Vm-1 and Vm-2 were fused using primers V1-F / R to obtain fusion fragment Vm;

[0099] The PCR reaction system described above is shown in Table 4, and the PCR reaction procedure is shown in Table 5.

[0100] The concentrations of HA3, HA4, MK, and Vm were determined, and ligation was performed using the Gibson assembly method. The ligation product was transformed into *Escherichia coli* DH10B competent cells and cultured at 37°C for 15 h on LB agar plates containing 50 μg / mL Apra and 50 μg / mL kanamycin sulfate (Kan). Subsequent plasmid extraction, enzyme digestion verification, and sequencing alignment confirmed the correct construction of the recombinant plasmid pTAMK. A schematic diagram of the recombinant plasmid pTAMK is shown below. gfp The sequence of plasmid pTAMK is SEQ ID NO:3.

[0101] 1.3 Construction of pTAG plasmid:

[0102] Using pTB203 (sequence shown in SEQ ID NO:15) as a template, primers INp-up-F / R and INp-down-F / R were used to amplify the upstream and downstream homologous arms HA5 and HA6 of the integration site, respectively;

[0103] pET-28a(+)- gfp Using the sequence shown in SEQ ID NO:43 as a template, amplification was performed using primers gfp-F / R. Figure 3 Integrated fragment GFP;

[0104] Using pTA plasmid as a template, the plasmid backbone fragment Vg-1 was amplified using primers V1-F and INg20-R (the bold underlined part in SEQ ID NO:35 corresponding to Vg-1), and the plasmid backbone fragment Vg-2 was amplified using primers IN20g-F (the bold underlined part in SEQ ID NO:34 corresponding to Vg-2) and V1-R. Using Vg-1 and Vg-2 as templates, the plasmid backbone fragments Vg-1 and Vg-2 were fused to obtain the fusion fragment Vg using primers V1-F / R.

[0105] The PCR reaction system described above is shown in Table 4, and the PCR reaction procedure is shown in Table 5.

[0106] The concentrations of HA5, HA6, GFP, and Vg were determined, and ligation was performed using the Gibson assembly method. The ligation product was transformed into *Escherichia coli* DH10B competent cells and cultured at 37°C for 15 h on LB agar plates containing 50 μg / mL Apra. Subsequent plasmid extraction, enzyme digestion verification, and sequencing alignment confirmed the correct construction of the recombinant plasmid pTAG. A schematic diagram of the recombinant plasmid pTAG is shown below. E. fergusonii The sequence of plasmid pTAG is SEQ ID NO:42.

[0107] 2. Construction of Escherichia coli donor strain:

[0108] 2.1 Loss of endogenous plasmid pEFER:

[0109] Using sodium dodecyl sulfate (SDS) culture, Escherichia coli (Fergusonia) was cultured. bla ATCC 35469) was inoculated into 5 mL LB broth and cultured at 37°C with shaking at 200 rpm for 18 h. 50 μL was then inoculated into 5 mL LB broth containing 0.05% SDS and cultured at 37°C with shaking at 200 rpm for 18 h. The culture was repeatedly passaged in LB broth and LB broth containing SDS. When the culture reached the 8th-9th generation, the bacterial culture was streaked onto a plate, and single colonies were selected. Amplification was performed using plasmid-specific primers KP-F and KP-R according to the reaction system in Table 4 and the reaction program in Table 5. The amplification results were detected by electrophoresis. No bands indicated that the strain EF1001, which had lost the endogenous plasmid pEFER (composed of SEQ ID NO: 1, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60 in the 5'-3' direction), was obtained.

[0110] 2.2 Drug resistance genes bla TEM-1B Knockout

[0111] 2.2.1 Preparation of EF1001 competent cells

[0112] Using an inoculation loop, pick strain EF1001 (preserved in glycerol at -80℃), streak it onto antibiotic-free LB medium, and incubate overnight at 37℃. Pick a single colony from the above plate and inoculate it into 5 mL of antibiotic-free LB broth, incubate at 37℃ and 200 rpm for 12 h.

[0113] The cultured bacterial suspension was inoculated into 100 mL of LB liquid medium at a ratio of 1:100 and incubated at 37°C and 200 rpm. When OD... 600 When the value reaches 0.6~0.8 (about 2 hours of culture), stop the culture, aliquot the cultured bacterial solution into 50 mL centrifuge tubes, and place them on ice for 10 minutes to stop growth.

[0114] Place the centrifuge tubes in a pre-cooled centrifuge (4°C), centrifuge at 4000 rpm for 10 min at 4°C, and discard the supernatant.

[0115] Add 15 mL of pre-cooled ddH2O to each centrifuge tube to resuspend the bacterial cells, centrifuge at 4000 rpm and 4℃ for 10 min, and discard the supernatant.

[0116] Each centrifuge tube was added 15 mL pre-cooled 10% glycerol, resuspended the bacteria at 4000 rpm, 4°C, centrifuged for 10 min, and the supernatant was discarded. This step was repeated once.

[0117] The bacteria were resuspended with 500 μL pre-cooled 10% glycerol, and the EF1001 competent cells were divided into 1.5 mL EP tubes, 40 μL per tube. After completion, they were quickly stored at -80°C.

[0118] 2.2.2, pEcCas plasmid into EF1001

[0119] The strain EF1001 competent cells were taken out from the -80°C refrigerator, placed on ice to melt, and 400 ng pEcCas plasmid was added to 40 μL competent cells, which were gently mixed with a gun head.

[0120] The mixture was transferred to a clean and pre-cooled 1 mm shock cup to avoid air bubbles, and the shock cup was wiped clean of surface moisture and placed in a shock tank. The shock parameters were set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time about 5 ms.

[0121] Immediately add 1 mL LB broth, mix by blowing and sucking with a gun head, then transfer to a 1.5 mL EP tube, and incubate at 37°C, 200 rpm for 1 h.

[0122] Centrifuge at 8000 rpm, room temperature for 2 min to collect all the bacteria, and spread on LB plates containing 50 μg / mL Kan, and incubate at 37°C overnight.

[0123] The suspected positive single colony was picked with a sterile gun head into 3 mL LB broth containing 50 μg / mL Kan for expansion culture, 1 μL of bacterial solution was taken as a template, and PCR was performed with pEcCas specific primers cas-F / R. The correctly identified positive colony was named EF1002.

[0124] 2.2.3, Preparation of EF1002 competent cells:

[0125] The strain EF1002 (-80°C glycerol preserved bacteria) was picked with a inoculation loop, streaked on LB plates containing 50 μg / mL Kan, and incubated at 37°C in a constant temperature incubator overnight. Single colonies were picked from the above plate and inoculated into 5 mL LB broth containing 50 μg / mL Kan, and incubated at 37°C, 200 rpm for 13 h.

[0126] The cultured bacteria liquid was inoculated into 100 mL LB broth containing 50 μg / mL Kan and 30 mM L-arabinose at a volume ratio of 1:100, and cultured at 37°C and 200 rpm. When the OD value reached 0.6-0.8 (about 2 h of culture), the culture was stopped, and the cultured bacteria liquid was aliquoted into 50 mL centrifuge tubes and placed on ice for 10 min to stop growth. 600 The centrifuge tubes were placed in a pre-cooled centrifuge (4°C), centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was discarded. 15 mL of pre-cooled ddH2O was added to each centrifuge tube to resuspend the bacteria, which was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was discarded. 15 mL of pre-cooled 10% glycerol was added to each centrifuge tube to resuspend the bacteria, which was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was discarded. This step was repeated once. The bacteria were resuspended with 500 μL of pre-cooled 10% glycerol, aliquoted into 1.5 mL EP tubes at 40 μL per tube, and stored at -80°C after completion.

[0127] The centrifuge tubes were placed in a pre-cooled centrifuge (4°C), centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was discarded. 15 mL of pre-cooled ddH2O was added to each centrifuge tube to resuspend the bacteria, which was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was discarded. 15 mL of pre-cooled 10% glycerol was added to each centrifuge tube to resuspend the bacteria, which was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was discarded. This step was repeated once. The bacteria were resuspended with 500 μL of pre-cooled 10% glycerol, aliquoted into 1.5 mL EP tubes at 40 μL per tube, and stored at -80°C after completion.

[0128] 2.2.4, pTAKB plasmid into EF1002 and knockout verification:

[0129] The EF1002 competent cells were taken out of the -80°C freezer, thawed on ice, and 400 ng of pTAKB was added to 40 μL of competent cells. The mixture was gently mixed with a gun tip.

[0130] The mixture was transferred to a clean and pre-cooled 1 mm shock cup to avoid air bubbles. The shock cup was wiped clean of surface moisture and placed in the shock slot. The shock parameters were set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time about 5 ms.

[0131] Immediately add 1 mL of LB broth, mix by blowing and sucking with a gun tip, and then transfer to a 1.5 mL EP tube. Incubate at 37°C and 200 rpm for 1 h. Centrifuge at 8000 rpm and room temperature for 2 min to collect all the bacteria, and spread on LB plates containing 50 μg / mL Kan and 50 μg / mL Apra. Incubate at 37°C overnight.

[0132] The suspected positive single colony was picked into 50 μL of LB broth containing 50 μg / mL Kan and 50 μg / mL Apra with a sterile gun tip for expansion culture. 1 μL of the bacterial liquid was used as a template for PCR verification with primers blaK-F and blaK-R (see Table 4 for PCR reaction system and Table 5 for PCR reaction program). The knockout strain was selected according to the size of the product, named EF1003. bla TEM-1B The knockout strain was selected according to the size of the product, named EF1003.

[0133] 2.2.5, Elimination of plasmid pTAKB

[0134] The above selected lacIq-pLpp-mCherry-KanR TEM-1B The knockout strain was transferred to 3 mL of LB liquid medium containing 50 μg / mL Kan and 30 mM rhamnose, and cultured at 37°C and 200 rpm for 14 h. A small amount of culture liquid was streaked on an LB plate containing 50 μg / mL Kan using a loop, and incubated at 37°C overnight.

[0135] A single colony was picked up with a sterile gun head and inoculated in 50 μL of LB broth containing 50 μg / mL Kan for expansion, and 1 μL of the bacterial liquid was used as a template for PCR verification using primers TA-F and TA-R (see Table 4 for the PCR reaction system and Table 5 for the PCR reaction procedure). No amplification product indicated that pTAKB was successfully eliminated, and the correct strain was named EF1004.

[0136] 2.3, lacI Fragment integration

[0137] 2.3.1, Preparation of EF1004 competence

[0138] The strain EF1004 was prepared into EF1004 competence according to the preparation method of EF1002 competence provided in step 2.2.3.

[0139] 2.3.2, Transformation of plasmid pTAMK into EF1004 and integration verification

[0140] The EF1004 competent cells were taken out from the -80°C refrigerator, melted on ice, and 400 ng of pTAMK was added to 40 μL of the competent cells, which were gently mixed with a gun head.

[0141] The mixture was transferred to a clean and pre-cooled 1 mm electroporation cup to avoid air bubbles, and the surface water of the electroporation cup was wiped off and placed in the electroporation tank for electroporation. The electroporation parameters were set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal electroporation time about 5 ms.

[0142] Immediately, 1 mL of LB broth was added, and the mixture was mixed by blowing and sucking with a gun head, and then transferred to a 1.5 mL EP tube, and incubated at 37°C and 200 rpm for 1 h. All the bacteria were collected by centrifugation at 8000 rpm and room temperature for 2 min, and spread on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apra, and incubated at 37°C overnight.

[0143] Suspected positive single colonies were picked up with a sterile pipette tip and cultured in 50 μL of LB broth containing 50 μg / mL Kan and 50 μg / mL Apra. One μL of the bacterial culture was used as a template for PCR verification using primers MK-F and MK-R. Products were screened based on size. -pLpp-mCherry-Km q lacI R The integrated strain.

[0144] 2.3.3. Plasmid pTAMK elimination

[0145] Referring to the method provided in 2.2.5, with -pLpp-mCherry-Km q lacI R Using the integrated strain as the target, a strain that successfully eliminated pTAMK was obtained and named EF2004.

[0146] 2.3.4 Elimination of plasmid pEcCas

[0147] The strain EF2004 obtained above was transferred to 3 mL of LB broth containing 15% sucrose and cultured at 37°C and 200 rpm for 13 h. A small amount of the culture was then streaked onto an LB plate containing 15% sucrose using an inoculation loop and incubated overnight at 37°C with the plate inverted.

[0148] A single colony was picked using a sterile pipette tip and cultured in 50 μL of LB broth. One μL of the culture was used as a template for PCR verification using primers CAS-F and CAS-R. No amplification product indicated successful elimination of pEcCas. This strain is EF:: pLpp-mCherry-Km q - lacI R .

[0149] 2.4 Verification:

[0150] Use an inoculation loop to pick up EF:: -pLpp-mCherry-Km q Figure 5 R (Preserved bacteria at -80℃ with glycerol) Streaked onto LB agar plates containing 50 μg / mL Kan and incubated overnight at 37℃. Single colonies were picked from the above plates and inoculated into 5 mL of LB broth containing 50 μg / mL Kan, and incubated at 37℃ and 200 rpm for 14–16 h.

[0151] Take 1 mL of bacterial solution, centrifuge at 8000 rpm for 2 min to collect the bacteria, resuspend with 1 mL of normal saline, centrifuge at 8000 rpm for 2 min, discard the supernatant, finally resuspend with 1 mL of normal saline, and observe the sample with the bacterial suspension.

[0152] Observe under bright field (BF), red fluorescence channel (mCherry) and green fluorescence channel (GFP), respectively, and the results are shown in lacI -pLpp-mCherry-Km q gfp R Under the red fluorescence channel, red light can be observed, proving that mCherry is successfully expressed.

[0153] 3、 gfp Construction of the labeled drug-resistant plasmid pTB203: gfp

[0154] 3.1, pTB203 plasmid extraction and purification:

[0155] 3.1.1, inoculate E. coli ECCRA-119 into LB broth containing 4 μg / mL meropenem (MEM), 37°C, 200 rpm, cultivate for 14-16 h, centrifuge at 9000 rpm for 2 min to collect the bacteria, and use the commercial BAC / PAC large plasmid extraction kit to extract the plasmid pTB203.

[0156] 3.1.2, transfer the above extract into DH10B competent cells, activate at 37°C, 200 rpm for 1 h, then centrifuge at 8000 rpm, room temperature for 2 min to collect all the bacteria, and plate on LB plates containing 4 μg / mL MEM, 37°C inverted overnight culture.

[0157] 3.1.3, use a sterile gun head to pick suspected positive single colonies into 4 mL of LB broth containing 4 μg / mL MEM for expansion culture, take 1 μL of bacterial solution as a template, and use pTB203 specific primers ndm-F / R for PCR, and the correct positive colonies are DH10B strains carrying only pTB203.

[0158] 3.1.4, centrifuge the above culture at 9000 rpm for 2 min to collect the bacteria, use the commercial BAC / PAC large plasmid extraction kit to extract the plasmid pTB203, determine the plasmid concentration, and determine it as a single band by nucleic acid electrophoresis, and store at -20°C.

[0159] 3.2, gfp Label integration: ​​

[0160] 3.2.1, Preparation of DH10B electrotransformation competence, refer to 2.2.1.

[0161] 3.2.2, Simultaneous electroporation of plasmid pEcCas and plasmid pTAG into DH10B competence, the electroporation parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal electroporation time is about 5 ms. Incubate overnight on LB plate containing 50 μg / mL Kan and 50 μg / mL Apra, pick green suspected positive single colony, and perform PCR verification using primers cas-F / R. Name the verified correct colony as EC01.

[0162] 3.2.3, Refer to 2.2.3, prepare electrotransformation competence EC01 under the condition of double-antibiotic 50 μg / mL Kan and 50 μg / mL Apra.

[0163] 3.2.4, Transform pTB203 into electrotransformation competence EC01, the electroporation parameters are set as follows: 2 mm electroporation cup, voltage 2.5 kV, resistance 200 Ω, capacitance 25 μF, and ideal electroporation time is about 5 ms. After activation in 1 mL LB broth without antibiotic for 1 h, collect all bacteria by centrifugation at 8000 rpm for 2 min, and incubate overnight at 37°C on LB plate containing 50 μg / mL Kan, 50 μg / mL Apra and 4 μg / mL MEM. Pick suspected positive single colony, and perform PCR verification using primers GFPi-F / R. According to the product size, screen successfully labeled pTB203, and refer to Figure 4 for the map. gfp .

[0164] 3.2.5, Refer to 2.3.3 and 2.3.4, sequentially eliminate plasmids pTAG and pEcCas from the above verified correct strain, and obtain DH10B strain carrying only pTB203: gfp .

[0165] 3.2.6, Extract plasmid pTB203: gfp , determine the concentration, and store at -20°C.

[0166] 3.3, Fluorescence microscope observation

[0167] Use a inoculating loop to pick the pTB203: Figure 6DH10B strain (glycerol preservation at -80℃) was streaked on LB plate containing 4 μg / mL MEM and incubated overnight at 37℃ in constant temperature incubator. Single colony was picked from the above plate and inoculated into 5 mL LB broth containing 4 μg / mL MEM and incubated at 37℃, 200 rpm for 14-16 h.

[0168] 1 mL bacterial solution was taken and centrifuged at 8000 rpm for 2 min to collect the bacterial body, which was resuspended in 1 mL normal saline, centrifuged at 8000 rpm for 2 min, the supernatant was discarded, and finally resuspended in 1 mL normal saline. The bacterial suspension was used to prepare samples for observation.

[0169] Observation was carried out under bright field (BF), red fluorescence channel (mCherry) and green fluorescence channel (GFP) respectively, and the results are shown in gfp DH10B strain carrying pTB203: gfp could be observed under green fluorescence channel, which proved that GFP was successfully expressed.

[0170] 4. Construction of conjugation transfer donor strain EFmg-sy

[0171] 4.1. Construction of pTB203: lacI -pLpp-mCherry-Km q lacI R :

[0172] Reference is made to step 2.2.1 to prepare the strain EF: -pLpp-mCherry-Km q gfp R pTB203: lacI was transformed into EF: -pLpp-mCherry-Km q Figure 7 R ​The competent cells were prepared using a 2 mm electroporation cuvette with the following parameters: voltage 2.5 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time of about 5 ms. The suspected positive single colonies were selected and verified by PCR using primers GFPi-F / R, and the verified colonies were named Escherichia fergusonii EFmg-sy, which was preserved in the China Center for Type Culture Collection (CCTCC) with the accession number CCTCC NO: M2025310, address Wuhan, China, Wuhan University, postcode 430072, and preservation date February 27, 2025.

[0173] 4.2, fluorescence microscope observation results

[0174] The conjugative transfer donor strain Escherichia fergusonii EFmg-sy (hereinafter referred to as conjugative transfer donor strain EFmg-sy) was picked up with a inoculation loop, streaked on an LB plate containing 4 μg / mL MEM and 50 μg / mL Kan, and incubated overnight at 37°C in a constant temperature incubator. Single colonies were picked from the above plate and inoculated into 5 mL of LB broth containing 4 μg / mL MEM and 50 μg / mL Kan, and incubated at 37°C, 200 rpm, for 14-16 h.

[0175] 1 mL of the bacterial solution was taken, centrifuged at 8000 rpm for 2 min to collect the bacterial cells, resuspended with 1 mL of normal saline, centrifuged at 8000 rpm for 2 min, discarded the supernatant, finally resuspended with 1 mL of normal saline, and the bacterial suspension was used as a sample for observation.

[0176] Observation was carried out under bright field (BF), red fluorescence channel (mCherry), and green fluorescence channel (GFP), respectively, and the results are shown in gfp The conjugative transfer donor strain EFmg-sy strain could observe fluorescence under the red fluorescence channel, but could not observe fluorescence under the green fluorescence channel, proving that mCherry on the chromosome was successfully expressed, and the expression of GFP on plasmid pTB203: gfp was successfully inhibited in the donor strain.

[0177] Example 2

[0178] 1. Construction of editing plasmid

[0179] 1.1-1.3 same as example 1;

[0180] 1.4, Construction of pTAGmcr plasmid:

[0181] INmcr-up-F, INmcr-up-R and INmcr-down-F, INmcr-down-R to amplify the homologous arms HA7 and HA8 of the integration site upstream and downstream, respectively, using pTB44P3 as the template;

[0182] GFP using primers gfp-F / R, using pET-28a(+)- gfp as the template; Figure 8

[0183] INmcr20-R to amplify the plasmid backbone fragment Vg-3, and INmcr20p-F and V1-R to amplify the plasmid backbone fragment Vg-4, using pTA plasmid as the template; using Vg-3 and Vg-4 as the templates, and using primers V1-F / R, a fusion fragment Vgmcr was obtained by fusion PCR;

[0184] The above PCR reaction system is shown in Table 4 in Example 1, and the PCR reaction program is shown in Table 5 in Example 1;

[0185] The concentrations of HA7, HA8, GFP and Vgmcr were determined, and the connection was performed by the method of Gibson assembly, and the connection product was transformed into E. coli DH10B competent cells, and cultured on LB plates containing 50 μg / mL Apra at 37°C for 12-18 h. Subsequently, the correct recombinant plasmid pTAGmcr was obtained by plasmid extraction, enzyme digestion verification and sequencing comparison, and the sequence was SEQ ID NO: 47, and the atlas was as shown in gfp .

[0186] 2, Construction of E. gergusonii donor strain same as step 2 in example 1;

[0187] 3, gfp Construction of labeled drug-resistant plasmid pTB44P3: gfp

[0188] 3.1, pTB44P3 plasmid extraction and purification

[0189] The E. fergusonii EF44 was inoculated into LB broth containing 2 μg / mL colistin (CL), and cultured at 37°C, 200 rpm for 14-16 h, centrifuged at 9000 rpm for 2 min, and the bacterial pellet was used to extract the plasmid pTB44P3 using a commercial BAC / PAC large plasmid extraction kit.​​

[0190] The above extract was transferred into DH10B competent cells, activated at 37°C, 200 rpm for 1 h, centrifuged at 8000 rpm, room temperature for 2 min to collect all the bacteria, and spread on LB plates containing 2 μg / mL CL, and incubated at 37°C overnight;

[0191] The suspected positive single colony was picked with a sterile gun head into 4 mL LB broth containing 2 μg / mL CL for expansion culture, 1 μL of the bacterial solution was used as a template for PCR with pTB44P3 specific primers mcr-F / R (PCR reaction system and reaction procedure are shown in Table 4 and Table 5 of Example 1), and the correct positive colony was identified as the DH10B strain carrying only pTB44P3.

[0192] The above cultured bacterial solution was centrifuged at 9000 rpm for 2 min to collect the bacteria, and the plasmid pTB44P3 was extracted using a commercial BAC / PAC large plasmid extraction kit, the plasmid concentration was determined, and it was determined to be a single band by nucleic acid electrophoresis, and was stored at -20°C.

[0193] 3.2、 gfp Marker integration

[0194] DH10B electrotransformation competent cells were prepared, and the method is described in Step 2.2.1 of Example 1.

[0195] pEcCas and pTAGmcr were simultaneously electrotransformed into DH10B competent cells, and the shock parameters were set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time about 5 ms. The overnight culture was selected on LB plates containing 50 μg / mL Kan and 50 μg / mL Apra, and the green suspected positive single colony was selected and verified by PCR with primers cas-F / R, and the verified correct colony was named EC02.

[0196] Referring to Step 2.2.3 of Example 1, electrotransformation competent cells EC02 were prepared under the condition of 50 μg / mL Kan and 50 μg / mL Apra;

[0197] The pTB44P3 was transformed into EC02, and the electric shock parameters were set as follows: 2 mm electric shock cup, voltage 2.5 kV, resistance 200 Ω, capacitance 25 μF, and ideal electric shock time about 5 ms. After activation in 1 mL of LB broth without antibiotics for 1 h, all bacteria were collected by centrifugation at 8000 rpm for 2 min, and then cultured overnight at 37°C on LB plates containing 50 μg / mL Kan, 50 μg / mL Apra and 2 μg / mL CL. The suspected positive single colonies were selected, and PCR verification was performed using primers GFPi-F / R. The successfully labeled pTB44P3 was screened according to the size of the product. gfp The successfully constructed pTB44P3: Figure 9 The plasmid is shown as follows. gfp

[0198] According to Reference Example 1, steps 2.3.3 and 2.3.4, the above-mentioned strain verified to be correct was used to eliminate the plasmids pTAGmcr and pEcCas in sequence, so as to obtain a DH10B strain carrying only pTB44P3: gfp .

[0199] According to Reference Example 1, step 3.1 (1), the pTB44P3: gfp was extracted, and the concentration was determined before being stored at -20°C.

[0200] 3.3, Fluorescence microscope observation

[0201] The DH10B strain carrying pTB44P3: Figure 10 (germ preservation bacteria at -80°C) was picked up with a inoculating loop, and streaked on an LB plate containing 2 μg / mL CL, and then cultured overnight at 37°C in a constant temperature incubator. Single colonies were picked up from the above-mentioned plate, inoculated into 5 mL of LB broth containing 2 μg / mL CL, and then cultured at 37°C and 200 rpm for 14-16 h.

[0202] 1 mL of bacterial solution was taken, centrifuged at 8000 rpm for 2 min to collect the bacteria, resuspended with 1 mL of normal saline, centrifuged at 8000 rpm for 2 min, discarded the supernatant, finally resuspended with 1 mL of normal saline, and then the bacterial suspension was used to prepare a sample for observation.

[0203] Observation was performed in the red fluorescence channel (mCherry) and the green fluorescence channel (GFP) respectively, and the results are shown as follows. gfp The DH10B strain carrying pTB44P3: gfp could be observed to emit green light in the green fluorescence channel, which proved that GFP was successfully expressed.

[0204] 4, Construction of EFmg-cj:

[0205] ​4.1, pTB44P3: lacI into EF: -pLpp-mCherry-Km q lacI R

[0206] Reference Example 1, Step 2.2.1, to prepare EF: -pLpp-mCherry-Km q gfp R electrocompetent; pTB44P3: lacI into EF: -pLpp-mCherry-Km q Figure 11 R competent, using a 2 mm cuvette with the following shock parameters: voltage 2.5 kV, resistance 200 Ω, capacitance 25 μF, and an ideal shock time of about 5 ms. The suspected positive single colony was selected and verified by PCR using primers GFPi-F / R, and the verified colony was named Escherichia fergusonii EFmg-cj, which was preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M20251730, a preservation address of Wuhan, China, Wuhan University, a zip code of 430072, and a preservation date of July 30, 2025.

[0207] 4.2, fluorescence microscope observation results

[0208] The Escherichia fergusonii EFmg-cj strain (referred to as EFmg-cj strain) was picked up with a inoculating loop (-80℃ glycerol preserved bacteria), streaked on an LB plate containing 2 μg / mL CL and 50 μg / mL Kan, and cultured overnight in a constant temperature incubator at 37℃. A single colony was picked up from the above plate and inoculated into 5 mL of LB broth containing 2 μg / mL CL and 50 μg / mL Kan, and cultured at 37℃, 200 rpm, for 14~16 h. 1 mL of the bacterial solution was centrifuged at 8000 rpm for 2 min to collect the bacterial cells, resuspended with 1 mL of normal saline, centrifuged at 8000 rpm for 2 min, discarded the supernatant, finally resuspended with 1 mL of normal saline, and the bacterial suspension was used to prepare samples for observation. Observation was carried out under the red fluorescence channel (mCherry) and the green fluorescence channel (GFP), respectively, and the results are shown in gfp The EFmg-cj strain can be observed under the red fluorescence channel, but cannot be observed under the green fluorescence channel, proving that the mCherry on the chromosome is successfully expressed, and the plasmid pTB44P3: gfpGFP expression was successfully suppressed in the donor bacteria.

[0209] Experimental Example

[0210] Take the pTB203:: carried by the sample obtained in step 3.2.5 of Example 1 respectively. Figure 12 The DH10B strain and the conjugation transfer donor strain EFmg-sy constructed in step 4 of Example 1 were centrifuged at 8000 rpm for 2 min to collect the bacterial cells. The cells were resuspended in 1 mL of physiological saline, centrifuged at 8000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in 1 mL of physiological saline. The bacterial suspension was used as a sample for observation under bright field (BF), red fluorescence channel (mCherry), and green fluorescence channel (GFP).

[0211] Using EFmg-sy as the conjugation transfer donor, and Escherichia coli C600, Escherichia coli J53, and chicken fecal microbiota as the conjugation transfer recipients, conjugation transfer experiments were conducted.

[0212] The conjugation transfer experiment was performed as follows: Conjugation transfer donor and recipient bacteria in logarithmic growth phase were mixed at a 1:1 volume ratio and inoculated at 37°C for 12 h in antibiotic-free LB medium. 1 mL of bacterial suspension was collected by centrifugation at 8000 rpm for 2 min, resuspended in 1 mL of physiological saline, centrifuged again at 8000 rpm for 2 min, the supernatant was discarded, and the suspension was resuspended in 1 mL of physiological saline. Samples were prepared using this bacterial suspension for observation. Results are as follows: ​ As shown, this demonstrates that the conjugation transfer donor bacterium EFmg-sy can transfer plasmids into other recipient bacteria, and that donor and recipient bacteria can be distinguished by different fluorescence levels.

[0213] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A conjugation transfer donor bacterium, characterized in that, The conjugation transfer donor bacteria is *Escherichia fergusonii* EFmg-sy, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M2025310. or, The conjugation transfer donor strain is *Escherichia fergusonii* EFmg-cj, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M20251730.

2. A conjugation transfer donor bacterium as described in claim 1, having any of the following uses: (a) Detecting the conjugation and transfer capabilities of donor and recipient bacteria; (ii) Differentiate between donor and recipient bacteria in conjugation transfer; (iii) Detecting the transmissibility of drug resistance genes; (iv) Assess the diversity of recipient bacteria.

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