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

Abstract

The invention relates to the technical field of engineering bacteria, in particular to conjugational transfer receptor bacteria and a construction method and application thereof. The invention provides a conjugational transfer receptor bacterium, and the conjugational transfer receptor bacterium is Escherichia fergusonii M112, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M20242751. The conjugational transfer acceptor bacterium provided by the invention is stable in character, is simple to operate as a conjugational transfer acceptor, has sodium azide resistance, and can be used as an acceptor bacterium to detect the conjugational transfer efficiency of donor bacterium plasmids under the screening condition of sodium azide.

Description

Technical Field

[0001] The present invention relates to the technical field of engineering bacteria, and in particular to a conjugative transfer receptor bacterium and a construction method and use thereof. Background Art

[0002] With the excessive, improper or abuse of antibiotics, the spread of drug-resistant genes has been promoted, bacterial resistance has been continuously enhanced, and the isolation rate of high-risk drug-resistant genes has increased, posing a serious threat to clinical treatment. Plasmid-mediated horizontal gene transfer is an important way for the spread of Gram-negative bacterial resistance genes. At present, the research on the horizontal transmission ability of plasmids mainly uses Escherichia coli J53 and C600 as recipient bacteria, which are of a single type.

[0003] Escherichia Fergusonii is a member of the genus Escherichia in the family Enterobacteriaceae. As a zoonotic conditional pathogen, it is widely present in food, the environment, and the intestines of humans and animals. Escherichia Fergusonii was first isolated from clinical samples, and was subsequently reported to be isolated from cases of urinary tract infection, wound infection, bacteremia, etc., mostly in patients with weakened or impaired immunity. Like other species of the family Enterobacteriaceae, Escherichia Fergusonii is multidrug-resistant. Its potential as a reservoir of resistance genes has been underestimated in the past. In recent years, more and more literature reports have reported multidrug-resistant Escherichia Ferguson isolated from animals and clinically, including some high-risk resistance genes such as mcr , bla NDM , tet (X4). Currently, the detection of the conjugative transfer ability of plasmids carrying drug-resistant genes in drug-resistant bacteria mainly uses Escherichia coli J53 (sodium azide resistance) and C600 (rifampicin resistance) as recipient bacteria, but there is a lack of evaluation of cross-species transmission of plasmids. In addition, the study of the transmission of drug-resistant plasmids in Escherichia Ferguson also lacks a research model for intraspecies transmission. Therefore, the construction of an effective Escherichia Ferguson conjugative transfer recipient strain provides a diverse research model for the study of horizontal transmission of drug-resistant genes. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of the lack of conjugation transfer recipient bacteria in the prior art, thereby providing a conjugation transfer recipient bacteria and a construction method and use thereof.

[0005] In one aspect, the present invention provides a conjugative transfer recipient bacterium, wherein the conjugative transfer recipient bacterium is Escherichia coli ( Escherichia fergusonii )M112, has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, Postal Code 430072, the deposit number is CCTCC NO: M 20242751, and the deposit date is December 6, 2024.

[0006] In some embodiments, the conjugative transfer recipient bacteria is Escherichia coli, which is synthesized by eliminating endogenous plasmids and knocking out drug resistance genes. bla TEM-1B , the 112th amino acid of mutant protein SecA was obtained.

[0007] In some embodiments, the endogenous plasmid comprises plasmid pEFER.

[0008] In some of the embodiments, the deposit number of the Escherichia fergusonii is ATCC 35469.

[0009] In some of the embodiments, the amino acid at position 112 of the mutant protein SecA is mutated from alanine to valine.

[0010] On the other hand, the present invention provides a method for constructing a conjugative transfer recipient bacterium, comprising the following steps: eliminating the endogenous plasmid of Escherichia Fergusonii; introducing the gene editing plasmid into Escherichia Fergusonii; constructing a knockout bla TEM-1B Gene plasmid; construct SecA protein 112 amino acid mutation plasmid; knock out bla TEM-1B Plasmids for knocking out genes in Escherichia ferguson bla TEM-1BB Gene; using the 112th amino acid mutation plasmid of protein SecA to mutate the 112th amino acid of protein SecA of Escherichia ferguson.

[0011] In some of the embodiments, the method for eliminating endogenous plasmids of Escherichia ferguson includes a chemical elimination method, and the specific steps include treating cells with a surfactant, subculturing, and obtaining a strain in which the endogenous plasmid is lost, wherein the surfactant includes sodium dodecyl sulfate, and the sequence of the primer for verifying the loss of the endogenous plasmid is shown in SEQ ID NO: 1 to SEQ ID NO: 2.

[0012] In some of the embodiments, the step of introducing the gene-editing plasmid into Escherichia ferguson includes introducing the gene-editing plasmid into Escherichia ferguson by electric shock, verifying that the gene-editing plasmid includes a pEcCas plasmid, and verifying that the sequence of the primer for introducing the gene-editing plasmid into Escherichia ferguson is shown in SEQ ID NO: 25 to SEQ ID NO: 26.

[0013] In some embodiments, the knockout bla TEM-1B Methods for plasmids of genes include amplifying the gene using Escherichia fergusonii as a template bla TEM-1B The upstream and downstream homology arms of the gene were amplified and knocked out using pTA plasmid as a template blaTEM-1B The first plasmid backbone fragment and the second plasmid backbone fragment of the gene are amplified bla TEM-1B The upstream homology arm, downstream homology arm, first plasmid backbone fragment and second plasmid backbone fragment of the gene were connected by Gibson assembly method, and the connection product was injected into Escherichia coli competent cells to obtain knockout bla TEM-1B The plasmid of the gene, the amplification bla TEM-1B The primer sequences of the upstream homology arms of the gene are shown in SEQ ID NO: 3 to SEQ ID NO: 4. bla TEM-1B The primer sequences of the downstream homology arms of the gene are shown in SEQ ID NO: 5 to SEQ ID NO: 6, and the amplification knockout bla TEM-1B The primer sequences of the first plasmid backbone fragment of the gene are as shown in SEQ ID NO: 7 and SEQ ID NO: 10, and the amplification knockout bla TEM-1B The primer sequences of the second plasmid backbone fragment of the gene are shown in SEQ ID NO:8 and SEQ ID NO:9.

[0014] In some embodiments, the method for constructing a plasmid with a mutation in amino acid position 112 of protein SecA comprises amplifying the mutated plasmid using Escherichia fergusonii as a template. secA The first upstream homology arm fragment and the second upstream homology fragment of the mutation site are secA The upstream first homology arm fragment and the upstream second homology fragment of the gene mutation site are fused to secA The upstream homology arm fragment of the gene mutation site.

[0015] In some embodiments, Escherichia fergusonii was used as a template for amplification. secA The homology arm fragment downstream of the mutation site.

[0016] In some embodiments, pTA plasmid was used as template to amplify secA The first plasmid backbone fragment at the mutation site.

[0017] In some embodiments, pTA plasmid was used as template to amplify secA The second plasmid backbone fragment at the mutation site.

[0018] In some embodiments, secA The upstream homology arm fragment and downstream homology arm fragment of the mutation site secA The first plasmid backbone fragment at the mutation site, secAThe second plasmid backbone fragment at the mutation site was connected by Gibson assembly, and the connection product was transferred into Escherichia coli competent cells to obtain a plasmid with a mutation at amino acid position 112 of protein SecA. secA The primer sequences of the first homology arm fragment upstream of the mutation site are shown in SEQ ID NO: 11 to SEQ ID NO: 12. secA The primer sequences of the second homology arm fragment upstream of the mutation site are shown in SEQ ID NO: 13 to SEQ ID NO: 14. secA The first homology arm fragment upstream of the mutation site and secA The primer sequences of the second homology arm fragment upstream of the mutation site are shown in SEQ ID NO: 11 to SEQ ID NO: 14. secA The primer sequences of the downstream homology arm fragments of the mutation site are shown in SEQ ID NO: 15 to SEQ ID NO: 16. secA The primer sequences of the first plasmid backbone fragment of the mutation site are shown in SEQ ID NO: 7 and SEQ ID NO: 18. secA The primer sequences for the second plasmid backbone fragment at the mutation site are shown in SEQ ID NO:9 and SEQ ID NO:17.

[0019] In some embodiments, the knockout bla TEM-1B Plasmids for knocking out genes in Escherichia ferguson bla TEM-1B The steps include knocking out the gene by electroporation bla TEM-1B Plasmid introduction of genes into Escherichia Ferguson, resistance screening, verification, and knockout elimination bla TEM-1B The plasmid of the gene, wherein the Escherichia Ferguson includes at least one of the Escherichia Ferguson with a point mutation at position 112 of the SecA gene or the Escherichia Ferguson with a gene editing plasmid, and the verification method is a PCR method, and resistance screening knockout bla TEM-1B The sequences of primers for verifying the introduction of the plasmid of the gene into Escherichia ferguson are shown in SEQ ID NO: 19 to SEQ ID NO: 20. bla TEM-1B The method for knockout knockout plasmids includes shaking the selected strain in a medium containing 40-60 μg / mL antibiotics for 12-14 hours, streaking on a medium containing 40-60 μg / mL antibiotics for at least 12 hours, and verifying the elimination of knockout knockout blaTEM-1B The primer sequences for gene verification are shown in SEQ ID NO: 21 to SEQ ID NO: 22.

[0020] In some embodiments, the step of using a protein SecA 112 amino acid mutant plasmid to mutate the 112 amino acid of the protein SecA of Escherichia Ferguson comprises introducing the protein SecA 112 amino acid mutant plasmid into the Escherichia Ferguson by electric shock, resistance screening, verification, and elimination of the protein SecA 112 amino acid mutant plasmid, wherein the Escherichia Ferguson includes a knockout bla TEM-1B The method comprises the steps of: culturing the resistance-screened strain in a culture medium containing an antibiotic for 12-14 hours, and ...

[0021] In some of the embodiments, the construction method also includes the step of eliminating the gene editing plasmid, and the step of eliminating the gene editing plasmid includes shaking culturing the strain introduced with the gene editing plasmid in a culture medium containing 10-20wt% sucrose for 12-14 hours, and streaking culturing in a culture medium containing 10-20wt% sucrose for at least 12 hours.

[0022] At the same time, the above-mentioned conjugation transfer recipient bacteria provided by the present invention or the conjugation transfer recipient bacteria constructed by the above-mentioned conjugation transfer recipient bacteria construction method has any of the following uses: (i) Preparation of antibacterial drugs; (ii) Preparation of biological products resistant to sodium azide; (iii) Detection of conjugation transfer efficiency of donor bacterial plasmids; (iv) Engineering bacteria for bacterial genetic manipulation.

[0023] The technical solution of the present invention has the following advantages: The present invention provides a conjugative transfer recipient bacterium, wherein the conjugative transfer recipient bacterium is Escherichia coli ( Escherichia fergusonii ) M112, which has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: M20242751. The conjugative transfer recipient bacteria provided by the present invention have stable properties, are easy to operate as a conjugative transfer recipient, and have sodium azide resistance. They can be used as recipient bacteria to detect the conjugative transfer efficiency of donor bacterial plasmids using sodium azide as a screening condition.

[0024] The conjugative transfer recipient bacteria provided by the present invention uses Escherichia fergusonii as the original strain, and transforms the wild type Escherichia fergusonii ATCC 35469 by the CRISPR-Cas9 method, thereby deleting the drug resistance gene on the genome. bla TEM-1B , a point mutation was made in the amino acid at position 112 of the SecA protein, and the endogenous plasmid carried by the wild type was eliminated, and a conjugative transfer recipient bacterium that was sensitive to antibiotics, resistant to sodium azide, and did not carry a plasmid was constructed, which enriched the types of conjugative transfer recipient bacteria and provided a new model for studying the horizontal transmission of plasmids in Escherichia ferguson.

[0025] Biological deposit information The Escherichia Ferguson provided by the present invention ( Escherichia fergusonii )M112, has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, Postal Code 430072, the deposit number is CCTCC NO: M20242751, and the deposit date is December 6, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 is a schematic diagram of the pTKAB plasmid in Example 1 of the present invention; Figure 2 is a schematic diagram of the pTAM112 plasmid in Example 1 of the present invention; Figure 3 It is a growth curve diagram of strains in each experimental group and each control group in Experimental Example 1 of the present invention; Figure 4 The present invention is experimental example 2 with Citrobacter freundii ( Citrobacter freundii BC73) as the donor bacteria and Escherichia coli J53 as the recipient bacteria; Figure 5 The present invention is experimental example 2 with Citrobacter freundii ( Citrobacter freundii BC73) as the donor bacteria and the strain M112 prepared in Example 1 of the present invention as the recipient bacteria; Figure 6 This is a diagram showing the efficiency of conjugation transfer in Experimental Example 2 of the present invention, using Escherichia coli ECCRA-119 as the donor bacterium and Escherichia coli J53 as the recipient bacterium; Figure 7 This is a diagram showing the efficiency of conjugation transfer in Experimental Example 2 of the present invention, using Escherichia coli ECCRA-119 as the donor bacterium and the strain M112 prepared in Example 1 of the present invention as the recipient bacterium. DETAILED DESCRIPTION The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0028] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0029] In the embodiment of the present invention, Escherichia Ferguson ( Escherichia fergusonii ) The deposit number is ATCC35469.

[0030] LB broth medium formula: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, add water to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 20 min.

[0031] LB solid medium formula: Add 1.5% agar powder to LB broth medium.

[0032] pEcCas plasmid, pTA plasmid, Escherichia coli J53, Citrobacter freundii ( Citrobacter freundii BC73), Escherichia coli ECCRA-119 are all stored in the laboratory of University of Science and Technology of China Hangzhou Institute of Advanced Studies. Among them, pEcCas plasmid is commercially available, pTA plasmid sequence is shown in sequence number SEQ ID NO: 29, Escherichia coli J53 whole genome sequence is shown in Genebank accession number CP028702, Escherichia coli ECCRA-119 whole genome sequence is shown in Genebank accession number CP029242~CP029245 (wherein CP029242 is the whole genome information of Escherichia coli ECCRA-119, and CP029243-CP029245 are the information of three plasmids contained in Escherichia coli ECCRA-119), Citrobacter freundii ( Citrobacter freundiiFor the complete genome sequence of Citrobacter freundii BC73, please refer to Genebank accession numbers CP117475~CP117478 (CP117475 is the complete genome sequence of Citrobacter freundii BC73, and CP117476-CP117478 are the three plasmid information contained in Citrobacter freundii BC73).

[0033] Example 1 This embodiment provides a method for constructing a conjugative transfer recipient bacterium, and the specific steps and parameters are as follows: 1. Loss of endogenous plasmid pEFER in Escherichia Ferguson Using the sodium dodecyl sulfate (SDS) culture method, the bacteria were inoculated into 5 mL of LB broth medium, and cultured at 37°C, 200 rpm, and shaking for 18 h. 50 μL was inoculated into 5 mL of 0.05% SDS LB broth, and cultured at 37°C, 200 rpm, and shaking for 18 h. The culture was repeatedly subcultured in LB broth and LB broth containing SDS. When the culture reached the 8th to 9th generation, the bacterial liquid was streaked on a plate, and a single colony was selected. The plasmid-specific primers KP-F and KP-R were used for amplification using the reaction system in Table 4 and the reaction program in Table 5. The amplification results were detected by electrophoresis. The absence of a band indicated that the strain EF1001 in which the endogenous plasmid pEFER of Escherichia Ferguson was lost was obtained.

[0034] 2. Editing plasmid construction 2.1、 bla TEM-1B Construction of knockout plasmid pTAKB Using the wild-type Escherichia fergusonii genome as a template, primers bla-up-F (the homology arm sequence contained in the primer is the bold underlined portion in SEQ ID NO.3 in Table 1), bla-up-R and bla-down-F (the homology arm sequence contained in the primer is the bold underlined portion in SEQ ID NO.5 in Table 1), bla-down-R were used to amplify bla TEM-1B The upstream and downstream homology arms HA1 and HA2; Using the pTA plasmid as a template, primers V1-F and bla20-R (the gRNA sequence contained in the primers is the bold underlined portion in SEQ ID NO.10 in Table 1) are used to amplify the plasmid backbone fragment Vbla-1, and primers bla20-F (the gRNA sequence contained in the primers is the bold underlined portion in SEQ ID NO.8 in Table 1) and V1-R are used to amplify the plasmid backbone fragment Vbla-2; The above PCR reaction system 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 were determined. They were connected by the DNA assembly technique (Gibson assembly) and the connected products were transformed into Escherichia coli DH10B competent cells and cultured on LB plates containing 50 μg / mL apramycin sulfate (Apra) at 37 °C for 15 h. Subsequently, plasmid extraction, restriction enzyme verification and sequencing were performed using a kit to obtain the correctly constructed recombinant plasmid pTAKB. The schematic diagram of the recombinant plasmid pTAKB is shown in Figure 1 , the sequence of plasmid pTAKB is SEQ ID NO:27.

[0035] 2.2 Construction of SecA (A112V) mutant plasmid pTAM112 Using the wild-type Escherichia fergusonii genome as template, primers secA -up1-F / R and secA -up2-F / R amplification secA The upstream homology arm fragments HA3-1 and HA3-2 of the mutation site; using HA3-1 and HA3-2 as templates, using primers secA -up1-F and secA -up2-R, the fusion fragment HA3 was obtained by PCR; Use primers secA -down-F / R amplification secA The downstream homology arm fragment HA4 of the mutation site; Using pTA plasmid as template, primers V1-F and secA 20-R (the gRNA sequence contained in the primer is the bold underlined portion of SEQ ID NO.18 in Table 1) amplifies the plasmid backbone fragment V112-1; Use primers secA 20-F (the gRNA sequence contained in the primer is the bold underlined portion in SEQ ID NO.17 in Table 1) and V1-R to amplify the plasmid backbone fragment V112-2; The above PCR reaction system is shown in Table 4, and the PCR reaction procedure is shown in Table 5; The concentrations of fragments HA3, HA4, V112-1, and V112-2 were determined, and the fragments were connected by Gibson assembly. The connected products were transformed into E. coli DH10B competent cells and cultured at 37°C for 15 h on LB plates containing 50 μg / mL Apra. Subsequently, plasmid extraction, restriction enzyme verification, and sequencing were performed using a kit to obtain the correctly constructed recombinant plasmid pTAM112. The map is shown in Figure 2As shown, the sequence of the recombinant plasmid pTAM112 is SEQ ID NO: 28.

[0036] 3. Introduction of gene editing plasmid 3.1. Preparation of EF1001 competent cells Use an inoculation loop to pick up strain EF1001 (-80 ℃ glycerol stock), streak it on non-resistant LB medium, and culture it in a constant temperature incubator at 37 ℃ overnight. Pick a single colony from the above plate and inoculate it into 5 mL non-resistant LB broth, and culture it at 37 ℃, 200 rpm for 12 hours.

[0037] The cultured bacterial suspension was inoculated into 100 mL LB liquid medium at a ratio of 1:100 and cultured at 37 °C and 200 rpm. 600 When the value reaches 0.6-0.8 (about 2 h of cultivation), stop the cultivation, divide the cultured bacterial solution into 50 mL centrifuge tubes, and place them on ice for 10 min to stop growth.

[0038] Place the centrifuge tube in a precooled centrifuge (4°C), centrifuge at 4000 rpm, 4°C, for 10 min, and discard the supernatant.

[0039] Add 15 mL of pre-cooled ddH2O to each centrifuge tube to resuspend the bacteria, centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant.

[0040] Add 15 mL of pre-cooled 10% glycerol to each centrifuge tube to resuspend the cells, centrifuge at 4000 rpm, 4°C for 10 min, and discard the supernatant. Repeat this step once more.

[0041] Resuspend the cells with 500 μL of pre-cooled 10% glycerol and dispense EF1001 competent cells into 1.5 mL EP tubes, 40 μL per tube. After completion, store them quickly at -80 °C.

[0042] 3.2. pEcCas plasmid was transformed into strain EF1001 Take out the competent cells of strain EF1001 from the -80 ℃ refrigerator, place them on ice to thaw, add 400 ng of pEcCas plasmid to 40 μL of competent cells, and mix gently with a pipette tip.

[0043] Transfer the mixture into a clean and pre-cooled 1 mm electric shock cup to avoid bubbles, wipe the surface of the electric shock cup clean and put it into the electric shock tank for electric shock. The electric shock parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electric shock time is about 5 ms.

[0044] Immediately add 1 mL of LB broth, pipette to mix well, transfer to a 1.5 mL EP tube, and culture at 37 °C, 200 rpm for 1 h.

[0045] All the bacteria were collected by centrifugation at 8000 rpm at room temperature for 2 min, spread on LB plates containing 50 μg / mL kanamycin sulfate (Kan), and cultured inverted at 37°C overnight.

[0046] The suspected positive single colony was picked up with a sterile pipette tip and transferred to 3 mL of LB broth containing 50 μg / mL Kan for expansion culture. 1 μL of the bacterial solution was taken as a template and PCR was performed using the pEcCas specific primers cas-F / R. The correctly identified positive colony was named EF1002.

[0047] 3.3. Preparation of competent strain EF1002 Use an inoculation loop to pick up strain EF1002 (-80℃ glycerol stock), streak it on a LB solid medium plate containing 50 μg / mL Kan antibiotic, and culture it overnight in a constant temperature incubator at 37℃. Pick a single colony from the above plate and inoculate it into 5 mL LB broth containing 50 μg / mL Kan, and culture it at 37℃, 200 rpm for 13h.

[0048] The cultured bacterial suspension was inoculated into 100 mL of LB liquid medium 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. 600 When the value reaches 0.6-0.8 (about 2 hours of cultivation), stop the cultivation, divide the cultured bacterial liquid into 50 mL centrifuge tubes, and place them on ice for 10 minutes to stop the growth.

[0049] Place the centrifuge tube in a precooled centrifuge (4 ℃), centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant. Add 15 mL of precooled ddH2O to each centrifuge tube to resuspend the bacteria, centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant. Add 15 mL of precooled 10% glycerol to each centrifuge tube to resuspend the bacteria, centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant. Repeat this step once more. Resuspend the bacteria with 500 μL of precooled 10% glycerol, and dispense the competent cells into 1.5 mL EP tubes, 40 μL per tube, and store them quickly at -80℃ after completion.

[0050] 4. Transformation of pTAKB plasmid into strain EF1002 and knockout verification 4.1. pTAKB plasmid transformed into strain Take out EF1002 competent cells from -80 ℃ refrigerator, put them on ice to thaw, add 400ng pTAKB to 40 μL competent cells, and mix gently with a pipette tip.

[0051] Transfer the mixture into a clean and pre-cooled 1 mm electric shock cup to avoid bubbles, wipe the surface of the electric shock cup clean and put it into the electric shock tank for electric shock. The electric shock parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electric shock time is about 5 ms.

[0052] Immediately add 1 mL of LB broth, mix well by pipetting with a pipette, and transfer to a 1.5 mL EP tube, and culture at 37°C, 200 rpm for 1 hour. Centrifuge at 8000 rpm for 2 min at room temperature to collect all the bacteria, spread them on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apra, and culture them upside down at 37°C overnight.

[0053] The suspected positive single colony was picked up with a sterile pipette tip and expanded in 50 μL LB broth containing 50 μg / mL Kan and 50 μg / mL Apra. 1 μL of bacterial solution was taken as a template and PCR verification was performed with primers blaK-F and blaK-R (PCR reaction system see Table 4, PCR reaction procedure see Table 5). The product size was screened. bla TEM-1B Knockout strains.

[0054] 4.2. Elimination of plasmid pTAKB The above filtered bla 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, 200 rpm for 14 h. A small amount of culture solution was streaked on an LB plate containing 50 μg / mL Kan using an inoculation loop, and cultured inverted at 37°C overnight.

[0055] Use a sterile pipette tip to pick up a single colony and expand it in 50 μL LB broth containing 50 μg / mL Kan. Take 1 μL of the bacterial solution as a template and perform PCR verification with primers TA-F and TA-R (see Table 4 for the PCR reaction system and Table 5 for the PCR reaction procedure). The absence of an amplified product indicates that pTAKB has been successfully eliminated, and the correctly identified strain was named EF1004.

[0056] 5. secA Gene 112 amino acid point mutation 5.1 Preparation of EF1004 competent cells Use strain EF1004 as the basic cell and prepare EF1004 competent cells according to the method in 3.3.

[0057] 5.2. Transformation of pTAM112 into EF1004 and mutation verification Take out EF1004 competent cells from the -80 ℃ refrigerator, place them on ice to thaw, add 300 ng pTAKB to 40 μL competent cells, and mix gently with a pipette tip.

[0058] Transfer the mixture into a clean and pre-cooled 1 mm electric shock cup to avoid bubbles, wipe the surface of the electric shock cup clean and put it into the electric shock tank for electric shock. The electric shock parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electric shock time is about 5 ms.

[0059] Immediately add 1 mL of LB broth, mix well by pipetting with a pipette, and transfer to a 1.5 mL EP tube, and culture at 37°C, 200 rpm for 1 hour. Centrifuge at 8000 rpm for 2 min at room temperature to collect all the bacteria, spread them on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apra, and culture them upside down at 37°C overnight.

[0060] Use a sterile pipette tip to pick up a suspected positive single colony and expand it in 50 μL LB broth containing 50 μg / mL Kan and 50 μg / mL Apra. Take 1 μL of the bacterial solution as a template and perform PCR with primers M112-F and M112-R. Sequence the sequencing products and screen the correct protein SecA A112V mutant strain based on the sequencing results, that is, the 112th amino acid of the SecA protein mutated from alanine to valine.

[0061] 5.3. Elimination of pTAM112 plasmid The SecA A112V mutant was used as the object, and the strain that successfully eliminated pTAM112 was obtained by referring to the method and related materials in 4.2, and was named EF1005.

[0062] 6. pEcCas plasmid elimination The strain EF1005 obtained above was transferred to 3 mL of LB broth containing 15% sucrose, and cultured at 37°C, 200 rpm for 13 h. A small amount of the culture solution was streaked on an LB plate containing 15% sucrose using an inoculation loop, and cultured inverted at 37°C overnight.

[0063] Use a sterile pipette tip to pick up a single colony and expand it in 50 μL LB broth. Take 1 μL of the bacterial solution as a template and perform PCR verification using primers cas-F and cas-R. If there is no amplification product, pEcCas has been successfully eliminated and the strain is M112.

[0064] The primer information used in this example 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 program is shown in Table 5.

[0065] Table 1 Primer information

[0066] Table 2 Strain information

[0067] Table 3 Plasmid information

[0068] Table 4 PCR reaction system

[0069] Table 5 PCR reaction program

[0070] Example 2 This embodiment provides a method for constructing a conjugative transfer recipient bacterium, and the specific steps and parameters are as follows: 1. Loss of endogenous plasmid pEFER in Escherichia Ferguson Using the sodium dodecyl sulfate (SDS) culture method, the bacteria were inoculated into 5 mL of LB broth medium, cultured at 37°C, 200 rpm for 16 h, 50 μL was inoculated into 5 mL of 0.05% SDS LB broth, cultured at 37°C, 200 rpm for 16 h, and repeatedly subcultured in LB broth and LB broth containing SDS. When cultured to the 8th to 9th generation, the bacterial liquid was streaked on a plate, and a single colony was selected. The plasmid-specific primers KP-F and KP-R were used for amplification, and the amplification results were detected by electrophoresis. The absence of a band indicated that the strain EF1001 in which the endogenous plasmid pEFER of Escherichia Ferguson was lost was obtained.

[0071] 2. Edit plasmid construction 2.1、 bla TEM-1B Construction of knockout plasmid pTAKB Using the wild-type Escherichia fergusonii genome as a template, primers bla-up-F (the homology arm sequence contained in the primer is the bold underlined portion in SEQ ID NO.3 in Table 1), bla-up-R and bla-down-F (the homology arm sequence contained in the primer is the bold underlined portion in SEQ ID NO.5 in Table 1), bla-down-R were used to amplify bla TEM-1B The upstream and downstream homology arms HA1 and HA2; Using the pTA plasmid as a template, primers V1-F and bla20-R (the gRNA sequence contained in the primers is the bold underlined portion in SEQ ID NO.10 in Table 1) are used to amplify the plasmid backbone fragment Vbla-1, and primers bla20-F (the gRNA sequence contained in the primers is the bold underlined portion in SEQ ID NO.8 in Table 1) and V1-R are used to amplify the plasmid backbone fragment Vbla-2; The above PCR reaction system 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 were determined. They were connected by the DNA assembly technique (Gibson assembly) and the connected products were transformed into Escherichia coli DH10B competent cells and cultured on LB plates containing 50 μg / mL apramycin sulfate (Apra) at 37 °C for 15 h. Subsequently, plasmid extraction, restriction enzyme verification and sequencing were performed using a kit to obtain the correctly constructed recombinant plasmid pTAKB. The schematic diagram of the recombinant plasmid pTAKB is shown in Figure 1 .

[0072] 2.2 Construction of SecA (A112V) mutant plasmid pTAM112 Using the wild-type Escherichia fergusonii genome as template, primers secA -up1-F / R and secA -up2-F / R amplification secA The upstream homology arm fragments HA3-1 and HA3-2 of the mutation site; using HA3-1 and HA3-2 as templates, using primers secA -up1-F and secA -up2-R, the fusion fragment HA3 was obtained by PCR; using primers secA -down-F / R amplification secA The downstream homology arm fragment HA4 of the mutation site; using pTA plasmid as template, primers V1-F and secA 20-R (the gRNA sequence contained in the primer is the bold underlined portion in SEQ ID NO.18 in Table 1) to amplify the plasmid backbone fragment V112-1; using primers secA 20-F (the gRNA sequence contained in the primer is the bold underlined portion in SEQ ID NO.17 in Table 1) and V1-R to amplify the plasmid backbone fragment V112-2; The above PCR reaction system is shown in Table 4, and the PCR reaction procedure is shown in Table 5; The concentrations of fragments HA3, HA4, V112-1 and V112-2 were determined, and the ligation products were connected by the Gibson assembly method. The ligation products were transformed into Escherichia coli DH10B competent cells and cultured on LB plates containing 50 μg / mL 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 pTAM112, as shown in the figure. Figure 2 shown.

[0073] 3. Introduction of gene editing plasmid 3.1. Preparation of EF1001 competent cells Use an inoculation loop to pick up strain EF1001 (-80 ℃ glycerol stock), streak it on non-resistant LB medium, and culture it in a constant temperature incubator at 37 ℃ overnight. Pick a single colony from the above plate, inoculate it into 5 mL of non-resistant LB broth, and culture it at 37 ℃, 200 rpm for 14 hours.

[0074] The cultured bacterial suspension was inoculated into 100 mL LB liquid medium at a ratio of 1:100 and cultured at 37 °C and 200 rpm. 600 When the value reaches 0.6~0.8 (about 2 h of cultivation), stop the cultivation, divide the cultured bacterial solution into 50 mL centrifuge tubes, and place them on ice for 10 minutes to stop growth. Place the centrifuge tubes in a pre-cooled centrifuge (4 ℃), centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant. Add 15 mL of pre-cooled ddH2O to each centrifuge tube to resuspend the bacteria, centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant. Add 15 mL of pre-cooled 10% glycerol to each centrifuge tube to resuspend the bacteria, centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant. Repeat this step once more. Resuspend the bacteria with 500 μL of pre-cooled 10% glycerol, divide the EF1001 competent cells into 1.5 mL EP tubes, 40 μL per tube, and quickly store them at -80 ℃ after completion.

[0075] 3.2. pEcCas plasmid was transformed into strain EF1001 Take out the competent cells of strain EF1001 from the -80 ℃ refrigerator, place them on ice to thaw, add 600 ng of pEcCas plasmid to 40 μL of competent cells, and mix gently with a pipette tip.

[0076] Transfer the mixture into a clean and pre-cooled 1 mm electroporation cup to avoid bubbles. Wipe the surface of the electroporation cup and put it into the electroporation tank for electroporation. The electroporation parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electroporation time is about 5 ms. Immediately add 1 mL LB broth, mix with a pipette tip, and transfer to a 1.5 mL EP tube. Incubate at 37°C, 200 rpm for 1 h. Centrifuge at 8000 rpm for 2 min at room temperature to collect all the bacteria, spread them on LB plates containing 50 μg / mL kanamycin sulfate (Kan), and invert and culture overnight at 37°C.

[0077] The suspected positive single colony was picked up with a sterile pipette tip and transferred to 3 mL of LB broth containing 40 μg / mL Kan for expansion culture. 1 μL of the bacterial solution was taken as a template and PCR was performed using the pEcCas specific primers cas-F / R. The correctly identified positive colony was named EF1002.

[0078] 3.3. Preparation of competent strain EF1002 Use an inoculation loop to pick up strain EF1002 (-80℃ glycerol stock), streak it on a LB solid medium plate containing 50 μg / mL Kan antibiotic, and culture it overnight in a constant temperature incubator at 37℃. Pick a single colony from the above plate and inoculate it into 5 mL LB broth containing 50 μg / mL Kan, and culture it at 37℃, 200 rpm for 13h.

[0079] The cultured bacterial suspension was inoculated into 100 mL of LB liquid medium 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. 600 When the value reaches 0.6-0.8 (about 2 hours of cultivation), stop the cultivation, divide the cultured bacterial liquid into 50 mL centrifuge tubes, and place them on ice for 10 minutes to stop the growth.

[0080] Place the centrifuge tube in a precooled centrifuge (4°C), centrifuge at 4000 rpm, 4°C for 10 min, and discard the supernatant.

[0081] Add 15 mL of pre-cooled ddH2O to each centrifuge tube to resuspend the bacteria, centrifuge at 4000 rpm, 4 ℃ for 10 min, and discard the supernatant.

[0082] Add 15 mL of pre-cooled 10% glycerol to each centrifuge tube to resuspend the cells, centrifuge at 4000 rpm, 4°C for 10 min, and discard the supernatant. Repeat this step once more.

[0083] Resuspend the bacteria with 500 μL of pre-cooled 10% glycerol, and dispense the competent cells into 1.5 mL EP tubes, 40 μL per tube. After completion, quickly store them at -80 ℃.

[0084] 4. secA Gene 112 amino acid point mutation 4.1. Transformation of pTAM112 into EF1002 and mutation verification Take out EF1002 competent cells from the -80℃ refrigerator, place them on ice to thaw, add 500ng pTAKB to 40 μL competent cells, and mix gently with a pipette tip.

[0085] Transfer the mixture into a clean and pre-cooled 1 mm electroporation cup to avoid bubbles. Wipe the surface of the electroporation cup and put it into the electroporation tank for electroporation. The electroporation parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electroporation time is about 5 ms. Immediately add 1 mL LB broth, blow and mix with a pipette tip, and transfer to a 1.5 mL EP tube. Incubate at 37 °C, 200 rpm for 1 hour. Centrifuge at 8000 rpm for 2 min at room temperature to collect all the bacteria, spread them on LB plates containing 50 μg / mL Kan and 50 μg / mL Apra, and invert and culture overnight at 37 °C.

[0086] Use a sterile pipette tip to pick up a suspected positive single colony and transfer it to 50 μL LB broth containing 50 μg / mL Kan and 50 μg / mL Apra for expansion culture. Take 1 μL of the bacterial solution as a template and perform PCR with primers M112-F and M112-R. Sequence the sequencing products and screen the correct protein SecA A112V mutant strain based on the sequencing results, that is, the 112th amino acid of the SecA protein mutated from alanine to valine.

[0087] 4.2. Elimination of pTAM112 plasmid The SecA A112V mutant was used as the object, and the method and related materials in 4.2 of Example 1 were referred to obtain a strain that successfully eliminated pTAM112, which was named EF1004.

[0088] 5. Transformation of pTAKB plasmid into strain EF1002 and knockout verification 5.1. pTAKB plasmid transformed into strain Use strain EF1004 as the basic cell and prepare EF1004 competent cells according to the method in 3.3.

[0089] Take out the EF1002 competent cells from the -80 ℃ refrigerator, place them on ice to thaw, add 400ng pTAKB to 40 μL competent cells, and mix gently with a pipette. Transfer the mixture to a clean and pre-cooled 1 mm electroporation cup to avoid bubbles, wipe the surface of the electroporation cup clean and put it into the electroporation tank for electroporation. The electroporation parameters are set as follows: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electroporation time is about 5 ms. Immediately add 1 mL LB broth, blow and mix with a pipette tip, and transfer to a 1.5mL EP tube, and culture at 37 ℃, 200 rpm for 1h. Centrifuge at 8000 rpm for 2 min at room temperature to collect all the bacteria, spread them on LB plates containing 50 μg / mL Kan and 50 μg / mL Apra, and culture them upside down overnight at 37 ℃.

[0090] The suspected positive single colony was picked up with a sterile pipette tip and expanded in 50 μL LB broth containing 50 μg / mL Kan and 50 μg / mL Apra. 1 μL of bacterial solution was taken as a template and PCR verification was performed with primers blaK-F and blaK-R (PCR reaction system see Table 4, PCR reaction procedure see Table 5). The product size was screened. bla TEM-1B Knockout strains.

[0091] 5.2. Elimination of plasmid pTAKB The above filtered bla 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, 200 rpm for 12 h. A small amount of culture solution was streaked on an LB plate containing 50 μg / mL Kan using an inoculation loop, and cultured inverted at 37°C overnight.

[0092] Use a sterile pipette tip to pick up a single colony and expand it in 50 μL LB broth containing 50 μg / mL Kan. Take 1 μL of the bacterial solution as a template and perform PCR verification with primers TA-F and TA-R (see Table 4 for the PCR reaction system and Table 5 for the PCR reaction procedure). The absence of an amplified product indicates that pTAKB has been successfully eliminated, and the correctly identified strain was named EF1005.

[0093] 6. pEcCas plasmid elimination The strain EF1005 obtained above was transferred to 3 mL of LB broth containing 10% sucrose, and cultured at 37°C, 200 rpm for 13 h. A small amount of the culture solution was streaked on an LB plate containing 10% sucrose using an inoculation loop, and cultured inverted at 37°C overnight.

[0094] Use a sterile pipette tip to pick up a single colony and expand it in 50 μL LB broth. Take 1 μL of the bacterial solution as a template and perform PCR verification using primers cas-F and cas-R. If there is no amplification product, pEcCas has been successfully eliminated and the strain is M112.

[0095] The primer information used in this example 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 program is shown in Table 5.

[0096] Experimental Example 1 The strain M112 prepared in Example 1 of the present invention was cultured in LB broth containing different concentrations of sodium azide, and the treatment groups with sodium azide concentrations of 0 μg / mL, 100 μg / mL, and 200 μg / mL were set as experimental group 1, experimental group 2, and experimental group 3, respectively.

[0097] Escherichia coli J53 was cultured in LB broth containing different concentrations of sodium azide, and the treatment groups with sodium azide concentrations of 0 μg / mL, 100 μg / mL, and 200 μg / mL were set as control group 1, control group 2, and control group 3, respectively.

[0098] The culture conditions of each experimental group and the control group were 37 ℃ and the culture time was 24 h.

[0099] according to Figure 3 It can be seen that the strain M112 provided by the present invention is resistant to sodium azide.

[0100] Experimental Example 2 Citrobacter freundii ( Citrobacter freundii BC73) and Escherichia coli ECCRA-119 were used as conjugative transfer donor bacteria, and the strain M112 obtained in Example 1 of the present invention and Escherichia coli J53 were used as conjugative transfer recipient bacteria. Citrobacter freundii BC73 Carry bla NDM-1 Gene, located on IncFIB plasmid, carried by conjugative transfer donor strain ECCRA-119 bla NDM-5 Gene, located on the IncX3 plasmid.

[0101] The donor bacteria and recipient bacteria in the logarithmic growth phase were mixed at a volume ratio of 1:1 and incubated in LB medium without resistance at 37°C for 8 h.

[0102] The mixed cultured bacteria were collected and spotted on LB plates containing sodium azide (100 μg / mL) (on which the recipient bacteria J53 and strain M112 could grow) and plates containing sodium azide (100 μg / mL) and meropenem (4 μg / mL) (on which the recipient bacteria that successfully obtained the plasmid, i.e., the conjugants, could grow). The conjugation transfer efficiency was calculated as the number of conjugants / the number of recipient bacteria.

[0103] Citrobacter freundii ( Citrobacter freundii BC73) is the donor bacterium. Figure 4 and Figure 5 The results of conjugation transfer using Escherichia coli ECCRA-119 as the donor bacterium are shown in Figure 6 and Figure 7 As shown ( Figure 6 and Figure 7 10 -1 ~10 -6 It indicates the dilution multiple of the bacterial solution after conjugation transfer. Specifically, after conjugation transfer, the bacteria are collected and resuspended in 1 mL of saline to form a bacterial solution. This bacterial solution is the original bacterial solution. The original bacterial solution is diluted 10 to 10 times. 6 Times 10 -1 ~10 -6 ). The conjugative transfer efficiency of the conjugative transfer recipient bacteria prepared in the embodiment of the present invention is at the same order of magnitude as that of Escherichia coli J53, and both have similar conjugative transfer efficiencies.

[0104] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A conjugative transfer recipient bacterium, characterized in that: The conjugative transfer recipient bacteria is Escherichia Ferguson ( Escherichia fergusonii )M112, has been deposited in China Center for Type Culture Collection with the deposit number CCTCCNO: M20242751.

2. The conjugative transfer recipient bacterium according to claim 1, characterized in that: The conjugative transfer recipient bacteria is Escherichia coli, which eliminates endogenous plasmids and knocks out drug resistance genes bla TEM-1B , the 112th amino acid of mutant protein SecA was obtained.

3. The conjugative transfer recipient bacterium according to claim 2, characterized in that: The endogenous plasmid comprises plasmid pEFER; and / or, The deposit number of the Escherichia Fergusonii is ATCC 35469; and / or, The 112th amino acid of the mutant protein SecA mutated from alanine to valine.

4. A method for constructing a conjugative transfer recipient bacterium according to any one of claims 1 to 3, characterized in that: The steps include: Elimination of endogenous plasmids of Escherichia fergusonii; Introducing gene-editing plasmids into Escherichia fergusonii; Construct knockout bla TEM-1B Plasmids containing genes; Constructing a plasmid for mutation of amino acid 112 of protein SecA; Knockout bla TEM-1B Plasmids for knocking out genes in Escherichia ferguson bla TEM-1B Gene; The 112th amino acid of protein SecA of Escherichia ferguson was mutated using the 112th amino acid mutation plasmid.

5. The construction method according to claim 4, characterized in that: The method for eliminating the endogenous plasmid of Escherichia fergusonii includes a chemical elimination method, and the specific steps include treating cells with a surfactant, subculturing, and obtaining a strain with lost endogenous plasmids. Wherein, the surfactant includes sodium dodecyl sulfate, The sequences of primers for verifying the loss of endogenous plasmids are shown in SEQ ID NO: 1 to SEQ ID NO: 2; and / or, The steps of introducing the gene editing plasmid into Escherichia Ferguson include introducing the gene editing plasmid into Escherichia Ferguson by electroporation, verifying that The gene editing plasmid includes a pEcCas plasmid, The sequences of primers for verifying the introduction of gene-edited plasmids into Escherichia ferguson are shown in SEQ ID NO: 25 to SEQ ID NO:

26.

6. The construction method according to claim 4, characterized in that: The knockout bla TEM-1B Methods for plasmids of genes include amplifying the gene using Escherichia fergusonii as a template bla TEM-1B The upstream and downstream homology arms of the gene were amplified and knocked out using pTA plasmid as a template bla TEM-1B The first plasmid backbone fragment and the second plasmid backbone fragment of the gene are amplified bla TEM-1B The upstream homology arm, downstream homology arm, first plasmid backbone fragment and second plasmid backbone fragment of the gene were connected by Gibson assembly method, and the connection product was injected into Escherichia coli competent cells to obtain knockout bla TEM-1B Gene plasmid, The amplification bla TEM-1B The primer sequences of the upstream homology arms of the gene are shown in SEQ ID NO: 3 to SEQ ID NO:

4. The amplification bla TEM-1B The primer sequences of the downstream homology arms of the gene are shown in SEQ ID NO: 5 to SEQ ID NO:

6. The amplification knockout bla TEM-1B The primer sequences of the first plasmid backbone fragment of the gene are as shown in SEQ ID NO: 7 and SEQ ID NO: 10, The amplification knockout bla TEM-1B The primer sequences of the second plasmid backbone fragment of the gene are shown in SEQ ID NO:8 and SEQ ID NO:

9.

7. The construction method according to claim 4, characterized in that: The method for constructing a SecA protein 112 amino acid mutation plasmid comprises amplifying the 112 amino acid mutation plasmid using Escherichia fergusonii as a template. secA The first upstream homology arm fragment and the second upstream homology fragment of the mutation site are secA The upstream first homology arm fragment and the upstream second homology fragment of the gene mutation site are fused to secA The upstream homology arm fragment of the gene mutation site; Amplification using Escherichia Ferguson as template secA The homology arm fragment downstream of the mutation site; Using pTA plasmid as template, amplify secA The first plasmid backbone fragment at the mutation site; Using pTA plasmid as template, amplify secA a second plasmid backbone fragment at the mutation site; Will secA The upstream homology arm fragment and downstream homology arm fragment of the mutation site secA The first plasmid backbone fragment at the mutation site, secA The second plasmid backbone fragment at the mutation site was connected by the Gibson assembly method, and the connection product was transferred into E. coli competent cells to obtain the 112th amino acid mutation plasmid of protein SecA. The amplification secA The primer sequences of the first homology arm fragment upstream of the mutation site are shown in SEQ ID NO: 11 to SEQ ID NO:

12. The amplification secA The primer sequences of the second homology arm fragment upstream of the mutation site are shown in SEQ ID NO: 13 to SEQ ID NO:

14. The fusion secA The first homology arm fragment upstream of the mutation site and secA The primer sequences of the second homology arm fragment upstream of the mutation site are shown in SEQ ID NO: 11 to SEQ ID NO:

14. The amplification secA The primer sequences of the downstream homology arm fragments of the mutation site are shown in SEQ ID NO: 15 to SEQ ID NO:

16. The amplification secA The primer sequences of the first plasmid backbone fragment of the mutation site are shown in SEQ ID NO: 7 and SEQ ID NO: 18, The amplification secA The primer sequences for the second plasmid backbone fragment at the mutation site are shown in SEQ ID NO:9 and SEQ ID NO:

17.

8. The construction method according to claim 4, characterized in that: Knockout bla TEM-1B Plasmids for knocking out genes in Escherichia ferguson bla TEM-1B The steps include knocking out the gene by electroporation bla TEM-1B Plasmid introduction of genes into Escherichia Ferguson, resistance screening, verification, and knockout elimination bla TEM-1B Plasmids containing genes, wherein Escherichia fergusonii includes secA At least one of Escherichia Fergusonii with a mutation in amino acid point 112 of the gene or Escherichia Fergusonii with a gene editing plasmid, Resistance screening knockout bla TEM-1B The sequences of primers for verifying the introduction of the plasmid of the gene into Escherichia fergusonii are shown in SEQ ID NO: 19 to SEQ ID NO:

20. The knockout bla TEM-1B The method for plasmid of the gene comprises shaking culturing the strain to be selected for resistance in a medium containing 40-60 μg / mL antibiotic for 12-14 hours, streaking culturing on a medium containing 40-60 μg / mL antibiotic for at least 12 hours, Validation of knockout elimination bla TEM-1B The primer sequences for gene verification are shown in SEQ ID NO: 21 to SEQ ID NO:

22.

9. The construction method according to claim 4, characterized in that: The step of using the 112th amino acid mutant plasmid of protein SecA to mutate the 112th amino acid of protein SecA of Escherichia Ferguson comprises introducing the 112th amino acid mutant plasmid of protein SecA into Escherichia Ferguson by electric shock method, and screening, verifying and eliminating the 112th amino acid mutant plasmid of protein SecA, wherein the Escherichia Ferguson comprises knocking out bla TEM-1B At least one of the following: Escherichia coli with a gene or Escherichia coli with a gene-editing plasmid, The verification primers for Escherichia fergusonii with a mutation in amino acid position 112 of the resistance screening protein SecA are shown in SEQ ID NO: 23 to SEQ ID NO:

24. The method for eliminating the 112th amino acid mutation plasmid of protein SecA comprises culturing the resistance-screened strain in a medium containing antibiotics for 12-14 hours, The verification primers for eliminating the 112th amino acid mutation plasmid of protein SecA are shown in SEQ ID NO: 25 to SEQ ID NO: 26; The construction method also includes the step of eliminating the gene editing plasmid, and the step of eliminating the gene editing plasmid includes shaking culturing the strain introduced with the gene editing plasmid in a culture medium containing 10-20wt% sucrose for 12-14 hours, and streaking culturing in a culture medium containing 10-20wt% sucrose for at least 12 hours.

10. A conjugation transfer recipient bacterium according to any one of claims 1 to 3 or a conjugation transfer recipient bacterium constructed by the construction method of a conjugation transfer recipient bacterium according to any one of claims 4 to 9 having any of the following uses: (1) Preparation of antibacterial drugs; (ii) Preparation of biological products resistant to sodium azide; (III) Detecting the conjugative transfer efficiency of the donor bacterial plasmid; (iv) Engineered bacteria used for bacterial genetic manipulation.

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