Joint transfer donor bacterium as well as construction method and application thereof

Abstract

The invention relates to the technical field of conjugational transfer engineering bacteria, in particular to a conjugational transfer donor bacterium as well as a construction method and application thereof, the conjugational transfer donor bacterium is Escherichia fergusonii EFmg-sy, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M2025310; the invention relates to a conjugational transfer donor bacterium which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M20251730, or the conjugational transfer donor bacterium is Escherichia fergusonii EFmg-cj and is preserved in China Center for Type Culture Collection (CCTCC). The conjugational metastasis donor bacterium provided by the invention is stable in character, is simple to operate as a conjugational metastasis donor, and can be used for rapidly detecting and distinguishing the conjugational metastasis donor bacterium and a recipient bacterium receiving plasmids through a fluorescence detection method, so that the detection of the plasmid horizontal transfer capability of the Escherichia fjosenii is realized.

Description

Technical Field

[0001] This invention relates to the field of conjugation transfer engineered bacteria technology, specifically to a conjugation transfer donor bacterium and its construction method and application. Background Technology

[0002] *Escherichia coli*, a member of the genus *Escherichia* in the family Enterobacteriaceae, is a zoonotic opportunistic pathogen widely distributed in the environment, food, and the intestines of humans and animals. Similar to other species in the Enterobacteriaceae family, *Escherichia coli* exhibits multidrug resistance. In recent years, an increasing number of studies have reported multidrug-resistant *Escherichia coli* isolates from animals and clinical cases, containing high-risk resistance genes for several important antibiotics, such as... mcr-1 , bla NDM and tet (X4) This poses a significant challenge to clinical treatment. Escherichia coli has a strong ability to accept exogenous plasmids with low adaptation costs, and carries the risk of spreading important drug resistance genes. Therefore, it is necessary to establish an efficient and reliable method to detect the ability of Escherichia coli to spread drug resistance plasmids.

[0003] Currently, the assessment of drug resistance gene transmission efficiency mainly relies on conjugation transfer experiments, with Escherichia coli J53 and C600 being commonly used recipient bacteria. This traditional detection method cannot simulate the conjugation transfer environment under natural conditions and cannot achieve high-throughput detection. Therefore, there is an urgent need to develop an efficient and sensitive method for detecting plasmid transmission ability, enabling a reliable assessment of the plasmid transmission ability of Escherichia coli under natural conditions, and providing data reference for the control of drug resistance gene transmission. Summary of the Invention

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

[0005] To this end, on the one hand, the present invention provides a conjugation transfer donor bacterium, wherein the conjugation transfer donor bacterium is Escherichia fergusonii EFmg-sy, which has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M2025310. Alternatively, the conjugation transfer donor bacterium is Escherichia fergusonii EFmg-cj, which has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M20251730.

[0006] On the other hand, 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. The endogenous plasmid pEFER of Escherichia coli was eliminated to obtain the first intermediate strain; Build bla TEM-1B The knockout strain specifically includes transferring plasmid pEcCas into the competent state of the first intermediate strain to form the second intermediate strain, transferring plasmid pTAKB into the competent state of the second intermediate strain to form the third intermediate strain, eliminating plasmid pTAKB in the third intermediate strain to form the fourth intermediate strain, and the gene sequence of plasmid pTAKB is shown in SEQ ID NO: 2. The plasmid pTAMK was transformed into the fourth intermediate strain, and the plasmids pTAMK and pEcCas were eliminated to form the target strain. The gene sequence of the plasmid pTAMK 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 in the 5'-3' direction, or 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.

[0007] In some embodiments, the sequences of primers used to verify the loss of the endogenous plasmid in the first intermediate strain are shown in SEQ ID NO: 5 to SEQ ID NO: 6.

[0008] In some of these embodiments, the knockout of the fourth intermediate strain is verified. bla TEM-1B The primer sequences for the gene are shown in SEQ ID NO: 7~SEQ ID NO: 8.

[0009] In some embodiments, the primer sequences for screening the fourth intermediate strain containing plasmid pTAMK are shown in SEQ ID NO: 9~SEQ ID NO: 10.

[0010] In some embodiments, the primer sequences used to verify the elimination of plasmid pTAMK by the third and / or fourth intermediate strains are shown in SEQ ID NO: 11 to SEQ ID NO: 12.

[0011] In some of these embodiments, the primer sequences used to verify the elimination of plasmid pEcCas by the fourth intermediate strain are shown in SEQ ID NO: 13~SEQ ID NO: 14.

[0012] In some embodiments, the method for constructing the target plasmid includes the following steps: using Escherichia coli ECCRA-119 as a template, extracting plasmid pTB203, transforming the extracted plasmid pTB203 into Escherichia coli DH10B, culturing and screening to obtain strains containing plasmid pTB203, extracting the plasmid to obtain plasmid pTB203, wherein the gene 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; Plasmids pEcCas and pTAG were transformed into Escherichia coli DH10B competent cells and screened to obtain the fifth intermediate strain. The gene sequence of plasmid pTAG is shown in SEQ ID NO: 42. The plasmid pTB203 was transferred into competent cells of the fifth intermediate strain, and after screening, plasmids pEcCas and pTAG were eliminated to obtain a strain containing the target plasmid, and the target plasmid was extracted.

[0013] In some embodiments, the method for constructing the target plasmid includes the following steps: using Escherichia coli EF44 as a template, extracting plasmid pTB44P3, transforming the extracted plasmid pTB44P3 into Escherichia coli DH10B, culturing and screening to obtain a strain containing plasmid pTB44P3, extracting the plasmid to obtain plasmid pTB44P3, wherein the gene sequence of plasmid pTB44P3 is composed 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; pEcCas and pTAGmcr were transformed into Escherichia coli DH10B competent cells and screened to obtain the sixth intermediate strain. The sequence of plasmid pTAGmcr is shown in SEQ ID NO:47. The plasmid pTB44P3 was transferred into the competent cells of the sixth intermediate strain. After screening, plasmids pEcCas and pTAGmcr were eliminated to obtain a strain containing the target plasmid. The target plasmid was then extracted.

[0014] In some embodiments, the primer sequences for verifying the presence of plasmid pTB203 in the strain containing plasmid pTB203 are shown in SEQ ID NO: 16~SEQ ID NO: 17.

[0015] In some of these embodiments, the primer sequences used to verify the transformation of the fifth intermediate strain into plasmid pEcCas are shown in SEQ ID NO: 13~SEQ ID NO: 14.

[0016] In some of these embodiments, the primer sequences used to verify the transformation of the fifth intermediate strain into plasmid pTB203 are shown in SEQ ID NO: 18~SEQ ID NO: 19.

[0017] In some embodiments, the primer sequences for verifying the elimination of plasmid pEcCas by strains containing the target plasmid are shown in SEQ ID NO: 13~SEQ ID NO: 14.

[0018] In some embodiments, the primer sequences for eliminating plasmid pTAG or plasmid pTB44P3 in strains containing the target plasmid are verified as shown in SEQ ID NO: 11~SEQ ID NO: 12.

[0019] In some embodiments, the primer sequences for verifying that the sixth intermediate strain contains plasmid pTB44P3 are shown in SEQ ID NO: 18~SEQ ID NO: 19.

[0020] In some embodiments, the method for constructing the plasmid pTAKB includes the following steps: using Escherichia coli Fergusonii as a template, amplifying the first upstream homologous arm using primers with sequences as shown in SEQ ID NO: 20~SEQ ID NO: 21, and amplifying the first downstream homologous arm using primers with sequences as shown in SEQ ID NO: 22~SEQ ID NO: 23; Using pTA plasmid as a template, the target plasmid backbone fragment was amplified using primers with sequences shown in SEQ ID NO: 24~SEQ ID NO: 25, and the second plasmid backbone fragment was amplified using primers with sequences shown in SEQ ID NO: 26~SEQ ID NO: 27. The first upstream homologous arm, the first downstream homologous arm, the target plasmid backbone fragment, and the second plasmid backbone fragment were connected and screened to obtain the plasmid pTAKB.

[0021] 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; 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(+)- mCherryThe sequence consists of SEQ ID NO: 46 and SEQ ID NO: 71 in the 5'-3' direction; 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. 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. 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.

[0022] 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. 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; 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. 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. 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.

[0023] 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; 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; Using pTA plasmid as a template, the target plasmid backbone fragment was amplified using primers with sequences as shown in SEQ ID NO: 24 and SEQ ID NO: 54, and the fifth plasmid backbone fragment was amplified using primers with sequences as shown in SEQ ID NO: 26 and SEQ ID NO: 53. Using the target plasmid backbone fragment and the fifth plasmid backbone fragment as templates, and primers with sequences as shown in SEQ ID NO: 24 and SEQ ID NO: 26, the target plasmid backbone fragment and the fifth plasmid backbone fragment were fused by fusion PCR to obtain the fusion fragment. The fourth upstream homologous arm, the fourth downstream homologous arm, gfp The integrated fragment and the fused fragment were connected, screened, and the plasmid pTAGmcr was obtained.

[0024] Meanwhile, the conjugation transfer donor bacteria or the conjugation transfer donor bacteria construction method provided by the present invention have any of the following uses: (a) Detecting the conjugation transfer ability 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.

[0025] The technical solution of this invention has the following advantages: 1. This invention provides a conjugation transfer donor bacterium, wherein the conjugation transfer donor bacterium is *Escherichia fergusonii* EFmg-sy, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2025310; or, the conjugation transfer donor bacterium is *Escherichia fergusonii* EFmg-cj, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20251730. The conjugation transfer donor bacterium provided by this invention is stable and simple to use as a conjugation transfer donor. It allows for rapid detection and differentiation of the conjugation transfer donor bacterium and the recipient bacterium that has received the plasmid using fluorescence detection, thereby enabling the detection of the horizontal plasmid transfer ability of *Escherichia fergusonii*.

[0026] 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.

[0027] 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.

[0028] 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

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of plasmid pTAKB in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of plasmid pTAMK in Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the plasmid pTAG in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the target plasmid in Embodiment 1 of the present invention; Figure 5 These are the fluorescence microscopy observation results of the target strain in Example 1 of this invention, with a scale bar of 20 μm; Figure 6 This is the fluorescence microscopy observation result of Escherichia coli DH10B containing the target plasmid in Example 1 of the present invention, with a scale bar of 20 μm; Figure 7 These are the fluorescence microscopy observation results of the conjugation transfer donor bacteria in Example 1 of this invention, with a scale bar of 20 μm; Figure 8 This is a spectrum of the pTAGmcr plasmid in Example 2 of the present invention; Figure 9 pTB44P3:: in Embodiment 2 of the present invention gfp Map of plasmids; Figure 10 The results of fluorescence microscopy observation in Example 2 of this invention are shown in section 3.3. Figure 11 This refers to the fluorescence microscopy observation results in Example 2 of the present invention, section 4.2. Figure 12 These are the fluorescence microscopy observation results of the transfer of conjugation donor bacteria to recipient bacteria in the experimental examples of this invention. Figure 12 The strain DH10B-pTB203-gfp, which expresses green fluorescent protein, is included in this study. Figure 12 The strain EFmg-sy, which expresses red fluorescent protein B, Figure 12 The "C" in the text indicates the conjugation result between strain EFmg-sy and the non-fluorescent recipient bacterium Escherichia coli C600. Figure 12 In this context, D represents the conjugation result of strain EFmg-sy and non-fluorescent Escherichia coli J53. Figure 12 In this context, E represents the conjugation result of strain EFmg and chicken manure bacteria on the cellulose filter membrane. Detailed Implementation The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0031] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0032] In this embodiment of the invention, the standard strain of Escherichia coli is Escherichia coli (Fergusonia). Escherichia fergusonii (The accession number is ATCC 35469).

[0033] The complete genome sequence of Escherichia coli ECCRA-119 can be found in GenBank accession numbers CP029242~CP029245 (where CP029242 is the complete genome information of Escherichia coli ECCRA-119, and CP029243-CP029245 are information on the three plasmids contained in Escherichia coli ECCRA-119).

[0034] Escherichia coli DH10B was purchased from Shanghai Sangon Biotech Co., Ltd.

[0035] Strain EF44 was disclosed in the literature by Tang B, Chang J, Chen Y, Lin J, Xiao X, Xia X, Lin J, Yang H, and Zhao G. Escherichia fergusonii, an underrated repository for antimicrobial resistance in food animals. Microbiology Spectrum. 2022, 10(1):e0161721.

[0036] 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.

[0037] Table 1 Primer Information

[0038] In Table 1, homologous arms are marked in italics and underlined, while gRNA is marked in bold underline.

[0039] Table 2. Strain Information

[0040] Table 3 Plasmid Information

[0041] Table 4 PCR reaction system

[0042] Table 5 PCR reaction procedure

[0043] Example 1 1. Construction of edit plasmids 1.1 Construction of pTAKB plasmid: Escherichia coli (Fergusonia) E. fergusonii 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. bla TEM-1B The upstream and downstream homologous arms HA1 and HA2; 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. The PCR reaction system described above is shown in Table 4, and the PCR reaction procedure is shown in Table 5. 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. Figure 1 The sequence of plasmid pTAKB is SEQ ID NO:2.

[0044] 1.2 Construction of pTAMK plasmid: Escherichia coli (Fergusonia) E. fergusonii 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. pET-28a(+)- mCherry Using mCherry-F / R primers as templates, amplification was performed. mCherr y integrated fragment MK ( lacI q -pLpp-mCherry-Km R ); 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; The PCR reaction system described above is shown in Table 4, and the PCR reaction procedure is shown in Table 5.

[0045] 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. Figure 2 The sequence of plasmid pTAMK is SEQ ID NO:3.

[0046] 1.3 Construction of pTAG plasmid: 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; pET-28a(+)- gfp Using the sequence shown in SEQ ID NO:43 as a template, amplification was performed using primers gfp-F / R. gfp Integrated fragment GFP; 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. The PCR reaction system described above is shown in Table 4, and the PCR reaction procedure is shown in Table 5. 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. Figure 3 The sequence of plasmid pTAG is SEQ ID NO:42.

[0047] 2. Construction of Escherichia coli donor strain: 2.1 Loss of endogenous plasmid pEFER: Using sodium dodecyl sulfate (SDS) culture, Escherichia coli (Fergusonia) was cultured. E. fergusonii 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.

[0048] 2.2 Drug resistance genes bla TEM-1B Knockout 2.2.1 Preparation of EF1001 competent cells 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Add 15 mL of pre-chilled 10% glycerol to each centrifuge tube to resuspend the bacterial cells. Centrifuge at 4000 rpm and 4°C for 10 min, then discard the supernatant. Repeat this step once more.

[0053] Resuspend the bacterial cells in 500 μL of pre-cooled 10% glycerol, and dispense 40 μL of EF1001 competent cells into 1.5 mL EP tubes. After completion, immediately store at -80°C.

[0054] 2.2.2 Transformation of pEcCas plasmid into EF1001 Remove competent cells of strain EF1001 from the -80℃ freezer, thaw them on ice, add 400 ng pEcCas plasmid to 40 μL of competent cells, and gently mix with a pipette tip.

[0055] Transfer the mixture into a clean, pre-cooled 1 mm shock cup, avoiding the formation of air bubbles. Wipe the surface of the shock cup dry and place it in the shock chamber for shock. The shock parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time approximately 5 ms.

[0056] Immediately add 1 mL of LB broth, mix well by pipetting, transfer to a 1.5 mL EP tube, and incubate at 37°C and 200 rpm for 1 h.

[0057] Collect all bacterial cells by centrifugation at 8000 rpm for 2 min at room temperature, spread them on LB plates containing 50 μg / mL Kan, and incubate overnight at 37°C with the plates inverted.

[0058] Suspected positive single colonies were picked up with a sterile pipette tip and cultured in 3 mL of LB broth containing 50 μg / mL Kan. 1 μL of the bacterial culture was used as a template and PCR was performed using the pEcCas specific primers cas-F / R. The correctly identified positive colonies were named EF1002.

[0059] 2.2.3 Preparation of EF1002 competent cells: Using an inoculation loop, pick strain EF1002 (preserved in glycerol at -80℃) and streak it onto an LB agar plate containing 50 μg / mL Kan. Incubate overnight at 37℃. Pick a single colony from the agar plate and inoculate it into 5 mL of LB broth containing 50 μg / mL Kan. Incubate at 37℃ and 200 rpm for 13 h.

[0060] The cultured bacterial suspension was inoculated at a volume ratio of 1:100 into 100 mL of LB broth containing 50 μg / mL Kan and 30 mM L-arabinose, 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 on ice for 10 minutes to stop growth.

[0061] Place the centrifuge tubes in a pre-chilled centrifuge (4°C) and centrifuge at 4000 rpm for 10 min, discarding the supernatant. Add 15 mL of pre-chilled ddH2O to each centrifuge tube to resuspend the bacterial cells, centrifuge at 4000 rpm for 10 min, discarding the supernatant. Add 15 mL of pre-chilled 10% glycerol to each centrifuge tube to resuspend the bacterial cells, centrifuge at 4000 rpm for 10 min, discarding the supernatant. Repeat this step once more. Resuspend the bacterial cells in 500 μL of pre-chilled 10% glycerol, aliquot 40 μL into 1.5 mL EP tubes, and immediately store at -80°C.

[0062] 2.2.4. pTAKB plasmid transformation into EF1002 and knockout verification: Remove EF1002 competent cells from the -80℃ freezer, thaw them on ice, add 400 ng pTAKB to 40 μL of competent cells, and gently mix with a pipette tip.

[0063] Transfer the mixture into a clean, pre-cooled 1 mm shock cup, avoiding the formation of air bubbles. Wipe the surface of the shock cup dry and place it in the shock chamber for shock. The shock parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time approximately 5 ms.

[0064] Immediately add 1 mL of LB broth, mix well by pipetting, and transfer to a 1.5 mL EP tube. Incubate at 37°C and 200 rpm for 1 h. Centrifuge at 8000 rpm for 2 min at room temperature to collect all bacterial cells. Spread the cells onto LB plates containing 50 μg / mL Kan and 50 μg / mL Apra, and incubate overnight at 37°C inverted.

[0065] 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 blaK-F and blaK-R (PCR reaction system is shown in Table 4, PCR reaction procedure is shown in 5). Products were screened based on size. bla TEM-1B The knockout strain was named EF1003.

[0066] 2.2.5. Plasmid pTAKB elimination The above-selected bla TEM-1B The knockout strains were 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 the culture was then streaked onto an LB plate containing 50 μg / mL Kan and incubated overnight at 37°C with the plate inverted.

[0067] A single colony was picked up with a sterile pipette tip and cultured in 50 μL of LB broth containing 50 μg / mL Kan. 1 μL of the bacterial culture was used as a template and PCR was performed using primers TA-F and TA-R for verification (PCR reaction system is shown in Table 4, PCR reaction procedure is shown in 5). No amplification product indicates that pTAKB was successfully eliminated, and the correctly identified strain was named EF1004.

[0068] 2.3 lacIq-pLpp-mCherry-KanR Fragment integration 2.3.1 Preparation of EF1004 competent cells Following the method for preparing EF1002 competent cells provided in step 2.2.3, strain EF1004 was prepared into EF1004 competent cells.

[0069] 2.3.2. Transformation of plasmid pTAMK into EF1004 and integration verification Remove EF1004 competent cells from the -80℃ freezer, thaw them on ice, add 400 ng pTAMK to 40 μL of competent cells, and gently mix with a pipette tip.

[0070] Transfer the mixture into a clean, pre-cooled 1 mm shock cup, avoiding the formation of air bubbles. Wipe the surface of the shock cup dry and place it in the shock chamber for shock. The shock parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal shock time approximately 5 ms.

[0071] Immediately add 1 mL of LB broth, mix well by pipetting, and transfer to a 1.5 mL EP tube. Incubate at 37°C and 200 rpm for 1 h. Centrifuge at 8000 rpm for 2 min at room temperature to collect all bacterial cells. Spread the cells onto LB plates containing 50 μg / mL Kan and 50 μg / mL Apra, and incubate overnight at 37°C inverted.

[0072] 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. lacI q -pLpp-mCherry-Km R The integrated strain.

[0073] 2.3.3. Plasmid pTAMK elimination Referring to the method provided in 2.2.5, with lacI q -pLpp-mCherry-Km RUsing the integrated strain as the target, a strain that successfully eliminated pTAMK was obtained and named EF2004.

[0074] 2.3.4 Elimination of plasmid pEcCas 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.

[0075] 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:: lacI q - pLpp-mCherry-Km R .

[0076] 2.4 Verification: Use an inoculation loop to pick up EF:: lacI q -pLpp-mCherry-Km 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.

[0077] Take 1 mL of bacterial suspension, centrifuge at 8000 rpm for 2 min to collect bacterial cells, resuspend in 1 mL of physiological saline, centrifuge at 8000 rpm for 2 min, discard the supernatant, and finally resuspend in 1 mL of physiological saline. Use this bacterial suspension as a sample for observation.

[0078] Observations were performed under bright field (BF), red fluorescence channel (mCherry), and green fluorescence channel (GFP) respectively, and the results are as follows: Figure 5 As shown, EF:: lacI q -pLpp-mCherry-Km R Red light was observed in the red fluorescence channel, proving that mCherry was successfully expressed.

[0079] 3. gfp The labeled drug resistance plasmid pTB203:: gfp Construction: 3.1 Extraction and purification of pTB203 plasmid: 3.1.1 Inoculate Escherichia coli ECCRA-119 into LB broth containing 4 μg / mL meropenem (MEM), incubate at 37℃ and 200 rpm for 14-16 h, centrifuge at 9000 rpm for 2 min, collect the bacterial cells, and extract plasmid pTB203 from the bacterial pellet using a commercial BAC / PAC large plasmid extraction kit.

[0080] 3.1.2. The above extract was transferred into DH10B competent cells, activated at 37°C and 200 rpm for 1 h, and then centrifuged at 8000 rpm at room temperature for 2 min to collect all cells. The cells were then spread on LB plates containing 4 μg / mL MEM and incubated overnight at 37°C with the plates inverted.

[0081] 3.1.3. Pick suspected positive single colonies with a sterile pipette tip and amplify them in 4 mL of LB broth containing 4 μg / mL MEM. Take 1 μL of the bacterial culture as a template and perform PCR using the pTB203-specific primer ndm-F / R. The correctly identified positive colonies are the DH10B strains that carry only pTB203.

[0082] 3.1.4. The above-mentioned bacterial culture solution was centrifuged at 9000 rpm for 2 min to collect the bacterial cells. The plasmid pTB203 was extracted using a commercial BAC / PAC large plasmid extraction kit. The plasmid concentration was determined and confirmed to be a single band by nucleic acid electrophoresis. The plasmid was then stored at -20℃.

[0083] 3.2 gfp Tag integration: 3.2.1 Preparation of DH10B electrocompetent states, the method is the same as in 2.2.1.

[0084] 3.2.2. Plasmids pEcCas and pTAG were simultaneously electroporated into DH10B competent cells. The electroporation parameters were set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal electroporation time approximately 5 ms. The cells were cultured overnight on LB agar plates containing 50 μg / mL Kan and 50 μg / mL Lapra. Single colonies showing a green color and suspected positive results were selected and verified by PCR using primers cas-F / R. The correctly verified colonies were named EC01.

[0085] 3.2.3. Referring to 2.2.3, prepare electrocompetent EC01 cells under the conditions of 50 μg / mL Kan and 50 μg / mL Apra dual antibodies.

[0086] 3.2.4. pTB203 cells were transferred into electrocompetent EC01 cells. Electroporation parameters were set as follows: 2 mm electroporation cuvette, 2.5 kV, 200 Ω, 25 μF, and ideal electroporation time of approximately 5 ms. After activation in 1 mL of antibiotic-free LB broth for 1 h, all cells were collected by centrifugation at 8000 rpm for 2 min and cultured overnight at 37°C on LB agar plates containing 50 μg / mL Kan, 50 μg / mL Apra, and 4 μg / mL MEM. Suspected positive single colonies were selected and verified by PCR using primers GFPi-F / R. Cells were screened based on product size. gfp The successfully labeled pTB203 is shown in the spectrum. Figure 4 .

[0087] 3.2.5. Referring to 2.3.3 and 2.3.4, the plasmids pTAG and pEcCas were sequentially removed from the correctly verified strains to obtain strains carrying only pTB203:: gfp The DH10B strain.

[0088] 3.2.6, Refer to 3.1 (1) Extract plasmid pTB203:: gfp After determining the concentration, store at -20℃.

[0089] 3.3 Fluorescence Microscopy Observation Use an inoculation loop to pick up the pTB203-carrying sample obtained in step 3.2.5. gfp The DH10B strain (preserved at -80℃ with glycerol) was streaked onto LB agar plates containing 4 μg / mL MEM and incubated overnight at 37℃. Single colonies were picked from the plates and inoculated into 5 mL of LB broth containing 4 μg / mL MEM, and incubated at 37℃ and 200 rpm for 14–16 h.

[0090] Take 1 mL of bacterial suspension, centrifuge at 8000 rpm for 2 min to collect bacterial cells, resuspend in 1 mL of physiological saline, centrifuge at 8000 rpm for 2 min, discard the supernatant, and finally resuspend in 1 mL of physiological saline. Use this bacterial suspension to prepare samples for observation.

[0091] Observations were performed under bright field (BF), red fluorescence channel (mCherry), and green fluorescence channel (GFP) respectively, and the results are as follows: Figure 6 As shown, carrying pTB203:: gfp The DH10B strain showed green light under the green fluorescence channel, proving that GFP was successfully expressed.

[0092] 4. Construction of conjugation transfer donor bacteria EFmg-sy 4.1, pTB203:: gfpTransplanted strain EF:: lacI q -pLpp-mCherry-Km R : Refer to step 2.2.1 and prepare strain EF:: lacI q -pLpp-mCherry-Km R To prepare competent states, pTB203:: gfp Transfer to EF:: lacI q -pLpp-mCherry-Km R Competent cells were cultured overnight on LB agar plates containing 50 μg / mL Kan and 4 μg / mL MEM. Single colonies suspected of being positive were selected and verified by PCR using primers GFPi-F / R. The colonies were named *Escherichia fergusonii* EFmg-sy, indicating that *Escherichia fergusonii* EFmg-sy was the conjugation transfer donor. The colonies were deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2025310, Wuhan University, Wuhan, China 430072, China, on February 27, 2025.

[0093] 4.2 Results of Fluorescence Microscopy Observation Using an inoculation loop, pick the conjugation transfer donor bacterium *Escherichia fergusonii* EFmg-sy (hereinafter referred to as the conjugation transfer donor strain EFmg-sy) (preserved in glycerol at -80℃), streak it onto an LB agar plate containing 4 μg / mL MEM and 50 μg / mL Kan, and incubate overnight at 37℃. Pick a single colony from the above agar plate and inoculate it into 5 mL of LB broth containing 4 μg / mL MEM and 50 μg / mL Kan, and incubate at 37℃ and 200 rpm for 14–16 h.

[0094] Take 1 mL of bacterial suspension, centrifuge at 8000 rpm for 2 min to collect bacterial cells, resuspend in 1 mL of physiological saline, centrifuge at 8000 rpm for 2 min, discard the supernatant, and finally resuspend in 1 mL of physiological saline. Use this bacterial suspension as a sample for observation.

[0095] Observations were performed under bright field (BF), red fluorescence channel (mCherry), and green fluorescence channel (GFP) respectively, and the results are as follows: Figure 7 As shown, the conjugation transfer donor strain EFmg-sy showed fluorescence under the red fluorescence channel, but not under the green fluorescence channel, proving that mCherry was successfully expressed on the chromosome, and that the plasmid pTB203:: gfp GFP expression was successfully suppressed in the donor bacteria.

[0096] Example 2 1. Construction of edit plasmids 1.1-1.3 are the same as in Example 1; 1.4 Construction of pTAGmcr plasmid: Using pTB44P3 as a template, primers INmcr-up-F, INmcr-up-R and INmcr-down-F, INmcr-down-R were used to amplify the upstream and downstream homologous arms HA7 and HA8 of the integration site, respectively. pET-28a(+)- gfp Using the template, amplification was performed using primers gfp-F / R. gfp Integrated fragment GFP; Using pTA plasmid as a template, the plasmid backbone fragment Vg-3 was amplified using primers V1-F and INmcr20-R, and the plasmid backbone fragment Vg-4 was amplified using primers INmcr20p-F and V1-R; using Vg-3 and Vg-4 as templates, the fusion fragment Vgmcr was obtained by fusion PCR using primers V1-F / R. The above PCR reaction system is shown in Table 4 of Example 1, and the PCR reaction procedure is shown in Table 5 of Example 1; The concentrations of HA7, HA8, GFP, and Vgmcr 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 12–18 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 pTAGmcr, with the sequence SEQ ID NO:47. (See diagram below.) Figure 8 As shown.

[0097] 2. The construction of the Escherichia coli donor strain was the same as step 2 in Example 1; 3. gfp The labeled drug resistance plasmid pTB44P3:: gfp Construction: 3.1 Extraction and purification of pTB44P3 plasmid Escherichia coli EF44 was inoculated into LB broth containing 2 μg / mL colistin (CL), incubated at 37°C and 200 rpm for 14–16 h, centrifuged at 9000 rpm for 2 min, and the bacterial cells were collected. The bacterial pellet was used to extract plasmid pTB44P3 using a commercial BAC / PAC large plasmid extraction kit. The above extract was transferred into DH10B competent cells, activated at 37°C and 200 rpm for 1 h, and all cells were collected by centrifugation at 8000 rpm and room temperature for 2 min. The cells were then spread on LB plates containing 2 μg / mL CL and incubated overnight at 37°C with the plates inverted. Suspected positive single colonies were picked up with a sterile pipette tip and cultured in 4 mL of LB broth containing 2 μg / mL CL. 1 μL of the bacterial culture was used as a template and PCR was performed using the pTB44P3 specific primers mcr-F / R (PCR reaction system and reaction procedure are shown in Tables 4 and 5 of Example 1). The correctly identified positive colonies were DH10B strains that carried only pTB44P3. The bacterial culture was centrifuged at 9000 rpm for 2 min to collect the bacterial cells. The plasmid pTB44P3 was extracted using a commercial BAC / PAC large plasmid extraction kit. The plasmid concentration was determined and confirmed to be a single band by nucleic acid electrophoresis. The plasmid was then stored at -20℃.

[0098] 3.2 gfp Tag integration The DH10B electrocompetent state was prepared according to step 2.2.1 of Example 1.

[0099] pEcCas and pTAGmcr were simultaneously electroporated into DH10B competent cells. The electroporation parameters were set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and ideal electroporation time of approximately 5 ms. The cells were cultured overnight on LB agar plates containing 50 μg / mL Kan and 50 μg / mL Apra. Single colonies showing a green color and suspected positive results were selected and verified by PCR using primers cas-F / R. The correctly verified colonies were named EC02. Following step 2.2.3 of Example 1, electrocompetent ECO2 cells were prepared under conditions of 50 μg / mL Kan and 50 μg / mL Apra bispecific antibodies; pTB44P3 cells were transferred to EC02 plate. Electroporation parameters were set as follows: 2 mm electroporation cuvette, 2.5 kV, 200 Ω, 25 μF, and ideal electroporation time of approximately 5 ms. After activation in 1 mL of antibiotic-free LB broth for 1 h, all cells were collected by centrifugation at 8000 rpm for 2 min and cultured overnight at 37°C on LB agar plates containing 50 μg / mL Kan, 50 μg / mL Apra, and 2 μg / mL CL. Suspected positive single colonies were selected and PCR was performed using primers GFPi-F / R for verification. Cells were screened based on product size. gfp pTB44P3 successfully tagged. pTB44P3:: successfully constructed. gfp plasmids such as Figure 9 As shown.

[0100] Referring to steps 2.3.3 and 2.3.4 of Example 1, the plasmids pTAGmcr and pEcCas were sequentially removed from the verified strain to obtain a strain carrying only pTB44P3:: gfp The DH10B strain.

[0101] Refer to step 3.1 (1) of Example 1 to extract pTB44P3:: gfp After determining the concentration, store at -20℃.

[0102] 3.3 Fluorescence Microscopy Observation Use an inoculation loop to pick up samples carrying pTB44P3:: gfp The DH10B strain (preserved at -80℃ with glycerol) was streaked onto LB agar plates containing 2 μg / mL Cl and incubated overnight at 37℃. Single colonies were picked from the plates and inoculated into 5 mL of LB broth containing 2 μg / mL Cl, and incubated at 37℃ and 200 rpm for 14–16 h.

[0103] Take 1 mL of bacterial suspension, centrifuge at 8000 rpm for 2 min to collect bacterial cells, resuspend in 1 mL of physiological saline, centrifuge at 8000 rpm for 2 min, discard the supernatant, and finally resuspend in 1 mL of physiological saline. Use this bacterial suspension to prepare samples for observation.

[0104] The results were observed under both the red fluorescence channel (mCherry) and the green fluorescence channel (GFP), as follows: Figure 10 As shown, carrying pTB44P3:: gfp The DH10B strain showed green light under the green fluorescence channel, proving that GFP was successfully expressed.

[0105] 4. Construction of EFmg-cj: 4.1, pTB44P3:: gfp Transfer to EF:: lacI q -pLpp-mCherry-Km R Referring to step 2.2.1 of Example 1, prepare EF:: lacI q -pLpp-mCherry-Km R Electrosensitive states; pTB44P3:: gfp Transfer to EF:: lacI q -pLpp-mCherry-Km R Competent cells were cultured overnight on LB agar plates containing 50 μg / mL Kan and 2 μg / mL Cl. Single colonies suspected of being positive were selected and verified by PCR using primers GFPi-F / R. The verified colonies were named *Escherichia fergusonii* EFmg-cj and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251730, Wuhan University, Wuhan, China 430072, China, on July 30, 2025.

[0106] 4.2 Results of Fluorescence Microscopy Observation Escherichia fergusonii EFmg-cj strain (abbreviated as EFmg-cj strain) (preserved in glycerol at -80℃) was picked using an inoculation loop and streaked onto an LB agar plate containing 2 μg / mL CL and 50 μg / mL Kan, and incubated overnight at 37℃. Single colonies were picked from the above agar plates and inoculated into 5 mL of LB broth containing 2 μg / mL CL and 50 μg / mL Kan, and incubated at 37℃ and 200 rpm for 14–16 h. 1 mL of bacterial suspension was collected by centrifugation at 8000 rpm for 2 min, resuspended in 1 mL of physiological saline, centrifuged 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. Observations were performed under the red fluorescence channel (mCherry) and the green fluorescence channel (GFP), respectively. The results are as follows: Figure 11 As shown, the EFmg-cj strain exhibits fluorescence under the red fluorescence channel but not under the green fluorescence channel, confirming successful expression of mCherry on the chromosome, and the plasmid pTB44P3:: gfp GFP expression was successfully suppressed in the donor bacteria.

[0107] Experimental Example Take the pTB203:: carried by the sample obtained in step 3.2.5 of Example 1 respectively. gfp 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).

[0108] 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.

[0109] 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: Figure 12 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.

[0110] 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 (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 method for constructing the conjugation transfer donor bacteria according to claim 1, characterized in that, Includes the following steps: The target plasmid was transferred into the target strain, whereby... The method for constructing the target strain includes the following steps: The endogenous plasmid pEFER of Escherichia coli was eliminated to obtain the first intermediate strain; Build bla TEM-1B The knockout strain specifically includes transferring plasmid pEcCas into the competent state of the first intermediate strain to form the second intermediate strain, transferring plasmid pTAKB into the competent state of the second intermediate strain to form the third intermediate strain, eliminating plasmid pTAKB in the third intermediate strain to form the fourth intermediate strain, and the gene sequence of plasmid pTAKB is shown in SEQ ID NO:

2. The plasmid pTAMK was transformed into the fourth intermediate strain, and the plasmids pTAMK and pEcCas were eliminated to form the target strain. The gene sequence of the plasmid pTAMK 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 in the 5'-3' direction, or 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.

3. The method for constructing conjugation transfer donor bacteria according to claim 2, characterized in that, The sequences of the primers used to verify the loss of the endogenous plasmid in the first intermediate strain are shown in SEQ ID NO: 5~SEQ ID NO: 6; Validation of the fourth intermediate strain knockout bla TEM-1B The primer sequences for the gene are shown in SEQ ID NO: 7~SEQ ID NO: 8; The primer sequences for screening the fourth intermediate strain containing plasmid pTAMK are shown in SEQ ID NO: 9~SEQ ID NO: 10; The primer sequences for verifying the elimination of plasmid pTAMK by the third and / or fourth intermediate strains are shown in SEQ ID NO: 11~SEQ ID NO: 12; The primer sequences for verifying the elimination of plasmid pEcCas by the fourth intermediate strain are shown in SEQ ID NO: 13~SEQ ID NO:

14.

4. The method for constructing the conjugation transfer donor bacteria according to claim 2, characterized in that, The method for constructing the target plasmid includes the following steps: Using Escherichia coli ECCRA-119 as a template, plasmid pTB203 was extracted and transformed into Escherichia coli DH10B. After cultivation and screening, strains containing plasmid pTB203 were obtained. Plasmid pTB203 was then extracted, and the gene 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. Plasmids pEcCas and pTAG were transformed into Escherichia coli DH10B competent cells and screened to obtain the fifth intermediate strain. The gene sequence of plasmid pTAG is shown in SEQ ID NO:

42. The plasmid pTB203 was transformed into competent cells of the fifth intermediate strain, and after screening, plasmids pEcCas and pTAG were eliminated to obtain strains containing the target plasmid. The target plasmid was then extracted; or, The method for constructing the target plasmid includes the following steps: Using Escherichia coli EF44 as a template, plasmid pTB44P3 was extracted and transformed into Escherichia coli DH10B. After cultivation and screening, strains containing plasmid pTB44P3 were obtained. Plasmid pTB44P3 was then extracted, and the gene sequence of plasmid pTB44P3 was composed 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. pEcCas and pTAGmcr were transformed into Escherichia coli DH10B competent cells and screened to obtain the sixth intermediate strain. The sequence of plasmid pTAGmcr is shown in SEQ ID NO:

47. The plasmid pTB44P3 was transferred into the competent cells of the sixth intermediate strain. After screening, plasmids pEcCas and pTAGmcr were eliminated to obtain a strain containing the target plasmid. The target plasmid was then extracted.

5. The method for constructing the conjugation transfer donor bacteria according to claim 4, characterized in that, The primer sequences for verifying the presence of plasmid pTB203 in the strain containing plasmid pTB203 are shown in SEQ ID NO: 16~SEQ ID NO: 17; The primer sequences for verifying the transformation of the fifth intermediate strain with plasmid pEcCas are shown in SEQ ID NO: 13~SEQ ID NO: 14; The primer sequences for verifying the transformation of the fifth intermediate strain with plasmid pTB203 are shown in SEQ ID NO: 18~SEQ ID NO: 19; The primer sequences for verifying the elimination of plasmid pEcCas by strains containing the target plasmid are shown in SEQ ID NO: 13~SEQ ID NO: 14; The primer sequences for eliminating plasmid pTAG or plasmid pTB44P3 in strains containing the target plasmid are shown in SEQ ID NO: 11~SEQ ID NO: 12; The primer sequences for verifying that the sixth intermediate strain contains plasmid pTB44P3 are shown in SEQ ID NO: 18~SEQ ID NO:

19.

6. The method for constructing conjugation transfer donor bacteria according to claim 2, characterized in that, The method for constructing the plasmid pTAKB includes the following steps. Using Escherichia coli Fergusoni as a template, the first upstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 20~SEQ ID NO: 21, and the first downstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 22~SEQ ID NO:

23. Using pTA plasmid as a template, the target plasmid backbone fragment was amplified using primers with sequences shown in SEQ ID NO: 24~SEQ ID NO: 25, and the second plasmid backbone fragment was amplified using primers with sequences shown in SEQ ID NO: 26~SEQ ID NO:

27. The first upstream homologous arm, the first downstream homologous arm, the target plasmid backbone fragment, and the second plasmid backbone fragment were connected and screened to obtain the plasmid pTAKB.

7. The method for constructing the conjugation transfer donor bacteria according to claim 2, characterized in that, The method for constructing the plasmid pTAMK includes the following steps. Using Escherichia coli Fergusoni as a template, the second upstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 28~SEQ ID NO: 29, and the second downstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 30~SEQ ID NO:

31. Using plasmid pET-28a(+)-mCherry as a template, primers with sequences shown in SEQ ID NO: 32~SEQ ID NO: 33 were used for amplification. lacI q -pLpp-mCherry-Km R The integrated fragment, plasmid pET-28a(+)-mCherry, consists of SEQ ID NO: 46 and SEQ ID NO: 71 in the 5'-3' direction; 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. 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. The second upstream homologous arm, the second upstream homologous arm, lacI q -pLpp-mCherry-Km R The integrated fragment and the fusion fragment were connected and screened to obtain the plasmid pTAMK. The sequence of plasmid pTAMK is shown in SEQ ID NO:

3.

8. The method for constructing conjugation transfer donor bacteria according to claim 2, characterized in that, The construction method of plasmid pTAG includes the following steps: Using plasmid pTB203 as a template, the third upstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 36~SEQ ID NO: 37, and the third downstream homologous arm was amplified using primers with sequences 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. 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; 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. 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. 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.

9. The method for constructing conjugation transfer donor bacteria according to claim 4, characterized in that, Includes the following steps: Using plasmid pTB44P3 as a template, the fourth upstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 49~SEQ ID NO: 50, and the fourth downstream homologous arm was amplified using primers with sequences shown in SEQ ID NO: 51~SEQ ID NO:

52. 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; Using pTA plasmid as a template, the target plasmid backbone fragment was amplified using primers with sequences shown in SEQ ID NO: 24 and SEQ ID NO: 54, and the fifth plasmid backbone fragment was amplified using primers with sequences shown in SEQ ID NO: 26 and SEQ ID NO:

53. Using the target plasmid backbone fragment and the fifth plasmid backbone fragment as templates, and primers with sequences as shown in SEQ ID NO: 24 and SEQ ID NO: 26, the target plasmid backbone fragment and the fifth plasmid backbone fragment were fused by fusion PCR to obtain the fusion fragment. The fourth upstream homologous arm, the fourth downstream homologous arm, gfp The integrated fragment and the fused fragment were connected, screened, and the plasmid pTAGmcr was obtained.

10. A conjugation transfer donor bacterium constructed using the method for constructing a conjugation transfer donor bacterium as described in claim 1 or any one of claims 2-9, having any one of the following uses: (a) Detecting the conjugation transfer ability 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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