A conjugation transfer recipient bacterium, a method for constructing the same, and uses thereof

Abstract

The present invention relates to the technical field of engineering bacteria, and particularly relates to a conjugation transfer recipient bacterium, a construction method thereof, and uses thereof. A conjugation transfer recipient bacterium provided by the present invention is Escherichia fergusonii ( Escherichia fergusonii ) M112, which has been deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M20242751. The conjugation transfer recipient bacterium provided by the present invention has stable traits, is simple to operate as a conjugation transfer recipient, and has sodium azide resistance, and can be used as a recipient bacterium to detect the conjugation 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 specifically relates to a conjugation transfer recipient bacterium, a construction method thereof, and uses thereof. Background Art

[0002] With the overuse, misuse, or abuse of antibiotics, the spread of drug-resistant genes has been promoted, leading to the continuous enhancement of bacterial drug resistance and the increase in the isolation rate of high-risk drug-resistant genes, posing a serious threat to clinical treatment. Plasmid-mediated horizontal gene transfer is an important route for the spread of drug-resistant genes in Gram-negative bacteria. Currently, the research on the horizontal transfer ability of plasmids mainly uses Escherichia coli J53 and C600 as recipient bacteria, with a single species.

[0003] Escherichia fergusonii is one of the members 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 subsequent reports have successively described its isolation from cases such as urinary tract infections, wound infections, and bacteremia, mostly in patients with low or impaired immune function. Escherichia fergusonii, like other species in the family Enterobacteriaceae, has multidrug resistance, and its potential as a reservoir of drug-resistant genes has been underestimated in the past. In recent years, more and more literature reports have described the isolation of multidrug-resistant Escherichia fergusonii from animals and clinically, including some high-risk drug-resistant genes such as mcr , bla NDM , tet (X4). Currently, for the detection of the conjugation transfer ability of drug-resistant gene-carrying plasmids in drug-resistant bacteria, Escherichia coli J53 (resistant to sodium azide) and C600 (resistant to rifampicin) are mainly used as recipient bacteria. However, there is a great lack of assessment of plasmid cross-species transmission. In addition, there is also a lack of a research model for the intraspecies transmission of Escherichia fergusonii drug-resistant plasmid transmission. Therefore, the construction of an effective Escherichia fergusonii conjugation transfer recipient strain provides diverse research models for the study of the 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 species of conjugation transfer recipient bacteria in the prior art, and thus provide a conjugation transfer recipient bacterium, a construction method thereof, and uses thereof.

[0005] On the one hand, the present invention provides a conjugation transfer recipient bacterium, which is Escherichia fergusonii ( Escherichia fergusonii ) M112, which has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan, China, Wuhan University, postal code 430072, deposit number CCTCC NO: M 20242751, and the deposit date being December 06, 2024.

[0006] In some of these embodiments, the conjugative transfer recipient bacterium is Escherichia fergusonii obtained by eliminating endogenous plasmids, knocking out drug resistance genes bla TEM-1B , and mutating the 112th amino acid of protein SecA.

[0007] In some of these embodiments, the endogenous plasmid includes plasmid pEFER.

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

[0009] In some of these embodiments, the 112th amino acid 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, including the following steps: eliminating the endogenous plasmid of Escherichia fergusonii; introducing a gene editing plasmid into Escherichia fergusonii; constructing a plasmid for knocking out bla TEM-1B a gene; constructing a plasmid with the 112th amino acid of protein SecA mutated; using the plasmid for knocking out bla TEM-1B the gene to knock out the bla TEM-1BB gene of Escherichia fergusonii; using the plasmid with the 112th amino acid of protein SecA mutated to mutate the 112th amino acid of protein SecA of Escherichia fergusonii.

[0011] In some of these embodiments, the method for eliminating the endogenous plasmid of Escherichia fergusonii includes using a chemical elimination method, and the specific steps include treating cells with a surfactant, subculturing, and obtaining a strain with the endogenous plasmid lost. Among them, the surfactant includes sodium dodecyl sulfate, and the sequences of the primers for verifying the loss of the endogenous plasmid are as shown in SEQ ID NO: 1 to SEQ ID NO: 2.

[0012] In some of these embodiments, the step of introducing the gene editing plasmid into Escherichia fergusonii includes introducing the gene editing plasmid into Escherichia fergusonii by electroporation. It is verified that the gene editing plasmid includes the pEcCas plasmid, and the sequences of the primers for verifying the introduction of the gene editing plasmid into Escherichia fergusonii are as shown in SEQ ID NO: 25 to SEQ ID NO: 26.

[0013] In some of these embodiments, the method for constructing the plasmid for knocking out bla TEM-1B the gene includes amplifying the upstream homologous arm and the downstream homologous arm of the bla TEM-1B gene using Escherichia fergusonii as a template, and amplifying the knockout blaTEM-1B The first plasmid backbone fragment and the second plasmid backbone fragment of the gene, and the amplified bla TEM-1B The upstream homologous arm, downstream homologous arm, first plasmid backbone fragment and second plasmid backbone fragment of the gene are ligated by the method of Gibson assembly, and the ligation product is injected into Escherichia coli competent cells to obtain the plasmid with the gene knocked out bla TEM-1B gene, and the primers for amplifying the bla TEM-1B upstream homologous arm of the gene are shown in SEQ ID NO: 3 to SEQ ID NO: 4, and the primers for amplifying the bla TEM-1B downstream homologous arm of the gene are shown in SEQ ID NO: 5 to SEQ ID NO: 6, and the primers for amplifying the knocked-out bla TEM-1B first plasmid backbone fragment of the gene are SEQ ID NO: 7 and SEQ ID NO: 10, and the primers for amplifying the knocked-out bla TEM-1B second plasmid backbone fragment of the gene are SEQ ID NO: 8 and SEQ ID NO: 9.

[0014] In some embodiments, the method for constructing the plasmid with the 112th amino acid of protein SecA mutated includes amplifying secA the upstream first homologous arm fragment and upstream second homologous fragment of the mutation site, and secA fusing the upstream first homologous arm fragment and upstream second homologous fragment of the gene mutation site into secA the upstream homologous arm fragment of the gene mutation site.

[0015] In some embodiments, amplifying secA the downstream homologous arm fragment of the mutation site using Escherichia fergusonii as a template.

[0016] In some embodiments, amplifying secA the first plasmid backbone fragment of the mutation site using the pTA plasmid as a template.

[0017] In some embodiments, amplifying secA the second plasmid backbone fragment of the mutation site using the pTA plasmid as a template.

[0018] In some embodiments, secA the upstream homologous arm fragment, downstream homologous arm fragment, secA the first plasmid backbone fragment of the mutation site, secAThe second plasmid backbone fragment of the mutation site is ligated by the method of Gibson assembly, and the ligation product is transferred into competent Escherichia coli cells to obtain a plasmid with a mutation at the 112th amino acid of protein SecA. The amplification secA The primer sequences of the first upstream homologous arm fragment 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 upstream homologous arm fragment of the mutation site are shown in SEQ ID NO: 13 to SEQ ID NO: 14. The fusion secA The first upstream homologous arm fragment of the mutation site and secA The primer sequences of the second upstream homologous arm fragment 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 homologous arm fragment 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 of the second plasmid backbone fragment of the mutation site are shown in SEQ ID NO: 9 and SEQ ID NO: 17.

[0019] In some of these embodiments, using knockout bla TEM-1B The steps of knocking out the gene in Escherichia fergusonii using a plasmid containing the knockout bla TEM-1B The steps of knocking out the gene include introducing the plasmid containing the knockout bla TEM-1B gene into Escherichia fergusonii by electroporation, resistance screening, verification, and elimination of the plasmid containing the knockout bla TEM-1B gene. Among them, Escherichia fergusonii includes at least one of Escherichia fergusonii with a point mutation at the 112th amino acid of the SecA gene or Escherichia fergusonii with a gene editing plasmid. The verification method is PCR. The verification primer sequences for introducing the plasmid containing the knockout bla TEM-1B gene into Escherichia fergusonii are shown in SEQ ID NO: 19 to SEQ ID NO: 20. The method for eliminating the plasmid containing the knockout bla TEM-1B gene includes culturing the strain obtained by resistance screening in a medium containing 40 - 60 μg / mL antibiotic by shaking for 12 - 14 h, streaking on a medium containing 40 - 60 μg / mL antibiotic for at least 12 h, and verifying the elimination. blaTEM-1B The primer sequences for gene verification are shown in SEQ ID NO: 21 to SEQ ID NO: 22.

[0020] In some of these embodiments, the step of mutating the 112th amino acid of protein SecA in Escherichia fergusonii using the plasmid for mutating the 112th amino acid of protein SecA includes introducing the plasmid for mutating the 112th amino acid of protein SecA into Escherichia fergusonii by electroporation, resistance screening, verification, and eliminating the plasmid for mutating the 112th amino acid of protein SecA, wherein the Escherichia fergusonii includes at least one of Escherichia fergusonii with bla TEM-1B the gene knocked out or Escherichia fergusonii with a gene editing plasmid. The verification is sequencing verification. The verification primers for screening Escherichia fergusonii with the 112th amino acid of protein SecA mutated are shown in SEQ ID NO: 23 to SEQ ID NO: 24. The method for eliminating the plasmid for mutating the 112th amino acid of protein SecA includes culturing the strain screened for resistance in a medium containing antibiotics for 12 - 14 h. The verification primers for eliminating the plasmid for mutating the 112th amino acid of protein SecA are shown in SEQ ID NO: 25 to SEQ ID NO: 26.

[0021] In some of these embodiments, the construction method further includes a step of eliminating the gene editing plasmid. The step of eliminating the gene editing plasmid includes culturing the strain into which the gene editing plasmid has been introduced in a medium containing 10 - 20 wt% sucrose by shaking for 12 - 14 h, and streaking the culture in a medium containing 10 - 20 wt% sucrose for at least 12 h.

[0022] Meanwhile, the above-mentioned conjugation transfer recipient bacterium provided by the present invention or the conjugation transfer recipient bacterium constructed by the construction method of the above-mentioned conjugation transfer recipient bacterium has any of the following uses:

[0023] (1) Preparing antibacterial drugs; (2) Preparing biological products resistant to sodium azide; (3) Detecting the conjugation transfer efficiency of donor bacterium plasmids; (4) Engineering bacteria for bacterial genetic manipulation.

[0024] The technical solution of the present invention has the following advantages:

[0025] A conjugation transfer recipient bacterium provided by the present invention, the conjugation transfer recipient bacterium is Escherichia fergusonii ( Escherichia fergusonii ) M112, which has been deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M20242751. The conjugation transfer recipient bacterium provided by the present invention has stable traits, is simple to operate as a conjugation transfer recipient, and has resistance to sodium azide, and can be used as a recipient bacterium to detect the conjugation transfer efficiency of donor bacterium plasmids with sodium azide as the screening condition.

[0026] The conjugation transfer recipient bacterium provided by the present invention uses Escherichia fergusonii as the original strain, and through the CRISPR-Cas9 method, the wild-type Escherichia fergusonii ATCC 35469 is modified. By deleting the drug resistance genes on the genome bla TEM-1B , performing a point mutation on the 112th amino acid of the SecA protein, and eliminating the endogenous plasmid carried by the wild type, an antibiotic-sensitive, sodium azide-resistant and plasmid-free conjugation transfer recipient bacterium is constructed, enriching the types of conjugation transfer recipient bacteria, and at the same time providing a new model for studying the plasmid-level transmission of Escherichia fergusonii.

[0027] Biological deposit information

[0028] The Escherichia fergusonii ( Escherichia fergusonii ) M112 provided by the present invention has been deposited in the China Center for Type Culture Collection. The deposit address is Wuhan, China, Wuhan University, postcode 430072, deposit number is CCTCC NO: M20242751, and the deposit date is December 06, 2024. Brief description of the drawings

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 It is a schematic diagram of the pTKAB plasmid in Example 1 of the present invention;

[0031] Figure 2 It is a schematic diagram of the pTAM112 plasmid in Example 1 of the present invention;

[0032] Figure 3 It is a growth curve diagram of the strains in each experimental group and each control group in Experimental Example 1 of the present invention;

[0033] Figure 4 It is an effect diagram of the conjugation transfer efficiency with Citrobacter freundii ( Citrobacter freundii BC73) as the donor bacterium and Escherichia coli J53 as the recipient bacterium in Experimental Example 2 of the present invention;

[0034] Figure 5 It is an effect diagram of the conjugation transfer efficiency with Citrobacter freundii ( Citrobacter freundii BC73) as the donor bacterium and the strain M112 prepared in Example 1 of the present invention as the recipient bacterium in Experimental Example 2 of the present invention;

[0035] Figure 6 It is the conjugation transfer efficiency effect diagram in Experimental Example 2 of the present invention, with Escherichia coli ECCRA-119 as the donor bacterium and Escherichia coli J53 as the recipient bacterium;

[0036] Figure 7 It is the conjugation transfer efficiency effect diagram in Experimental Example 2 of the present invention, with 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 implementation manners

[0037] The following embodiments are provided to better further understand the present invention. They are not limited to the described optimal implementation manner, and do not constitute a limitation on 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 those of other prior arts falls within the protection scope of the present invention.

[0038] For those embodiments where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0039] Escherichia fergusonii in the embodiments of the present invention ( Escherichia fergusonii ) has a preservation number of ATCC35469.

[0040] LB broth medium formula: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, add water to 1 L, adjust the pH to 7.0, and sterilize by moist heat at 121 °C for 20 min.

[0041] LB solid medium formula: Add 1.5% agar powder to the LB broth medium.

[0042] pEcCas plasmid, pTA plasmid, Escherichia coli J53, Citrobacter freundii ( Citrobacter freundiiBC73) and Escherichia coli ECCRA-119 were both preserved in the laboratory of Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences. Among them, the pEcCas plasmid is commercially available. The sequence of the pTA plasmid is shown in SEQ ID NO:29. The complete genome sequence of Escherichia coli J53 is shown in Genebank accession number CP028702, and the complete genome sequence of Escherichia coli ECCRA-119 is shown in Genebank accession numbers CP029242 - CP029245 (where CP029242 is the complete genome information of Escherichia coli ECCRA-119, and CP029243 - CP029245 are the information of three plasmids contained in Escherichia coli ECCRA-119). The complete genome sequence of Citrobacter freundii ( Citrobacter freundii BC73) is shown in Genebank accession numbers CP117475 - CP117478 (where CP117475 is the complete genome sequence of Citrobacter freundii BC73, and CP117476 - CP117478 are the information of three plasmids contained in Citrobacter freundii BC73).

[0043] Example 1

[0044] This example provides a method for constructing a conjugation transfer recipient bacterium. The specific steps and parameters are as follows:

[0045] 1. Loss of the endogenous plasmid pEFER of Escherichia fergusonii

[0046] Using the sodium dodecyl sulfate (SDS) culture method, inoculate the bacteria into 5 mL of LB broth medium, shake culture at 37 °C and 200 rpm for 18 h. Take 50 μL and inoculate it into 5 mL of LB broth containing 0.05% SDS, shake culture at 37 °C and 200 rpm for 18 h. Repeatedly passage in LB broth and LB broth containing SDS. When culturing to the 8 - 9th generation, streak the bacterial solution on a plate, select single colonies, and amplify using plasmid-specific primers KP-F and KP-R with the reaction system in Table 4 and the reaction program in Table 5. Detect the amplification result by electrophoresis. The absence of a band indicates obtaining the strain EF1001 with the loss of the endogenous plasmid pEFER of Escherichia fergusonii.

[0047] 2. Construction of the editing plasmid

[0048] 2.1 bla TEM-1B Construction of the knockout plasmid pTAKB

[0049] Using the wild-type Escherichia fergusonii genome as a template, primers bla-up-F (the homologous arm sequence contained in the primer is the thick underlined part in SEQ ID NO.3 in Table 1), bla-up-R and bla-down-F (the homologous arm sequence contained in the primer is the thick underlined part in SEQ ID NO.5 in Table 1), bla-down-R were used to amplify bla TEM-1B the upstream and downstream homologous arms HA1 and HA2;

[0050] Using the pTA plasmid as a template, primers V1-F and bla20-R (the gRNA sequence contained in the primer is the thick underlined part in SEQ ID NO.10 in Table 1) were used to amplify the plasmid backbone fragment Vbla-1, and primers bla20-F (the gRNA sequence contained in the primer is the thick underlined part in SEQ ID NO.8 in Table 1) and V1-R were used to amplify the plasmid backbone fragment Vbla-2;

[0051] The above PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5;

[0052] The above four fragments were recovered and their concentrations were measured, and they were ligated by the method of DNA assembly technology (Gibson assembly). The ligation product was transformed into Escherichia coli DH10B competent cells and cultured on an LB plate containing 50 μg / mL apramycin sulfate (Apra) at 37 °C for 15 h. Subsequently, plasmid extraction, restriction enzyme digestion verification and sequencing alignment were carried out through a kit to obtain the correctly constructed recombinant plasmid pTAKB. The schematic diagram of the recombinant plasmid pTAKB is shown in Figure 1 and the sequence of plasmid pTAKB is SEQ ID NO:27.

[0053] 2.2 Construction of SecA (A112V) mutant plasmid pTAM112

[0054] Using the wild-type Escherichia fergusonii genome as a template, primers secA -up1-F / R and secA -up2-F / R were used to amplify secA the upstream homologous arm fragments HA3-1 and HA3-2 of the mutation site; Using HA3-1 and HA3-2 as templates, primers secA -up1-F and secA -up2-R were used to obtain the fusion fragment HA3 by PCR;

[0055] Primers secA -down-F / R were used to amplify secAThe downstream homologous arm fragment HA4 of the mutation site;

[0056] Using the pTA plasmid as a template, and using primers V1-F and secA 20-R (the gRNA sequence contained in the primer is the thick underlined part of SEQ ID NO.18 in Table 1) to amplify the plasmid backbone fragment V112-1;

[0057] Using the primer secA 20-F (the gRNA sequence contained in the primer is the thick underlined part of SEQ ID NO.17 in Table 1) and V1-R to amplify the plasmid backbone fragment V112-2;

[0058] The above PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5;

[0059] Measure the concentrations of fragments HA3, HA4, V112-1 and V112-2, perform ligation by the method of Gibson assembly, transform the ligation product into Escherichia coli DH10B competent cells, and culture on an LB plate containing 50 μg / mL Apra at 37 °C for 15 h. Subsequently, plasmid extraction, enzyme digestion verification and sequencing alignment are carried out through a kit to obtain the correctly constructed recombinant plasmid pTAM112, and the map is as Figure 2 shown, and the sequence of the recombinant plasmid pTAM112 is SEQ ID NO: 28.

[0060] 3. Introduction of the gene editing plasmid

[0061] 3.1 Preparation of EF1001 competent

[0062] Use an inoculation loop to pick the strain EF1001 (glycerol-preserved bacteria at -80 °C), streak it on a non-resistant LB medium, and culture it overnight at 37 °C in a constant temperature incubator. Pick a single colony on the above plate and inoculate it into 5 mL of non-resistant LB broth, and culture at 37 °C and 200 rpm for 12 h.

[0063] Inoculate the cultured bacterial liquid into 100 mL of LB liquid medium at a ratio of 1:100, and culture at 37 °C and 200 rpm. When the OD 600 value reaches 0.6 - 0.8 (about 2 h of culture), stop the culture. Aliquot the cultured bacterial liquid into 50 mL centrifuge tubes and place them on ice for 10 min to stop growth.

[0064] Place the centrifuge tubes in a pre-cooled centrifuge (4 °C), centrifuge at 4000 rpm at 4 °C for 10 min, and discard the supernatant.

[0065] Add 15 mL of pre-cooled ddH 2The 0-fold resuspended thallus was centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was discarded.

[0066] Add 15 mL of pre-cooled 10% glycerol to each centrifuge tube to resuspend the thallus, centrifuge at 4000 rpm for 10 min at 4 °C, and discard the supernatant. This step was repeated once more.

[0067] Resuspend the thallus with 500 μL of pre-cooled 10% glycerol, aliquot the EF1001 competent cells into 1.5 mL EP tubes, 40 μL per tube, and quickly store at -80 °C after completion.

[0068] 3.2 Transformation of pEcCas plasmid into strain EF1001

[0069] Take out the EF1001 competent cells of the strain from the -80 °C refrigerator, place them on ice to melt, add 400 ng of pEcCas plasmid to 40 μL of competent cells, and gently mix with a pipette tip.

[0070] Transfer the mixture into a clean and pre-cooled 1 mm electroporation cuvette, avoid generating bubbles, wipe the surface moisture of the electroporation cuvette and put it into the electroporation chamber for electroporation. The electroporation parameters were set as: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electroporation time was about 5 ms.

[0071] Immediately add 1 mL of LB broth, mix well by pipetting and transfer to a 1.5 mL EP tube, and culture at 37 °C and 200 rpm for 1 h.

[0072] Centrifuge at 8000 rpm for 2 min at room temperature to collect all the thalli, spread them on an LB plate containing 50 μg / mL kanamycin sulfate (Kan), and incubate inverted at 37 °C overnight.

[0073] Pick the suspected positive single colony with a sterile pipette tip into 3 mL of LB broth containing 50 μg / mL Kan for amplification culture. Take 1 μL of the bacterial solution as a template and perform PCR with pEcCas-specific primers cas-F / R. Name the correctly identified positive colony EF1002.

[0074] 3.3 Preparation of competent cells of strain EF1002

[0075] Pick strain EF1002 (preserved in glycerol at -80 °C) with an inoculation loop, streak on an LB solid medium plate containing 50 μg / mL Kan antibiotic, and incubate overnight at 37 °C in a constant temperature incubator. Pick a single colony from the above plate and inoculate it into 5 mL of LB broth containing 50 μg / mL Kan, and culture at 37 °C and 200 rpm for 13 h.

[0076] Inoculate the cultured bacterial solution 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 culture it at 37 °C under the condition of 200 rpm. When the OD 600 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 min to stop growth.

[0077] Place the centrifuge tubes into a pre-cooled centrifuge (4 °C), centrifuge at 4000 rpm, 4 °C for 10 min, and discard the supernatant. Add 15 mL of pre-cooled ddH 2 O to resuspend the cells, centrifuge at 4000 rpm, 4 °C for 10 min, and discard the supernatant. 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. Resuspend the cells with 500 μL of pre-cooled 10% glycerol, aliquot the competent cells into 1.5 mL EP tubes, 40 μL per tube, and quickly store them at -80 °C after completion.

[0078] 4. Transformation of pTAKB plasmid into strain EF1002 and knockout verification

[0079] 4.1 Transformation of pTAKB plasmid into the strain

[0080] Take out the EF1002 competent cells from the -80 °C refrigerator, thaw them on ice, add 400 ng of pTAKB to 40 μL of competent cells, and gently mix with a pipette tip.

[0081] Transfer the mixture into a clean and pre-cooled 1 mm electroporation cuvette, avoid generating bubbles, wipe the surface moisture of the electroporation cuvette and place it in the electroporation chamber 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.

[0082] Immediately add 1 mL of LB broth, mix well by pipetting and transfer it to a 1.5 mL EP tube, culture it at 37 °C, 200 rpm for 1 h. Centrifuge at 8000 rpm at room temperature for 2 min to collect all the cells, spread them on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apramycin, and incubate it upside down at 37 °C overnight.

[0083] Pick the suspected positive single colony with a sterile pipette tip and transfer it to 50 μL of LB broth containing 50 μg / mL Kan and 50 μg / mL Apra for expanded culture. Take 1 μL of the bacterial solution as a template and perform PCR verification with primers blaK-F and blaK-R (the PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5). Screen according to the product size bla TEM-1B of the knockout strain.

[0084] 4.2 Elimination of plasmid pTAKB

[0085] Transfer the above-screened bla TEM-1B knockout strain to 3 mL of LB liquid medium containing 50 μg / mL Kan and 30 mM rhamnose, culture at 37 °C and 200 rpm for 14 h. Use an inoculation loop to pick up a small amount of the cultured bacterial solution and streak it on an LB plate containing 50 μg / mL Kan, and incubate it upside down at 37 °C overnight.

[0086] Pick a single colony with a sterile pipette tip and transfer it to 50 μL of LB broth containing 50 μg / mL Kan for expanded culture. Take 1 μL of the bacterial solution as a template and perform PCR verification with primers TA-F and TA-R (the PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5). The absence of amplification products indicates the successful elimination of pTAKB, and the strain with correct identification is named EF1004.

[0087] 5. secA Point mutation at the 112th amino acid of the gene

[0088] 5.1 Preparation of EF1004 competent cells

[0089] Using strain EF1004 as the basic cell, prepare EF1004 competent cells according to the method in 3.3.

[0090] 5.2 Transformation of pTAM112 into EF1004 and mutation verification

[0091] Take out the EF1004 competent cells from the -80 °C refrigerator, place them on ice to melt, add 300 ng of pTAKB to 40 μL of the competent cells, and gently mix with a pipette tip.

[0092] Transfer the mixture into a clean and pre-cooled 1 mm electroporation cuvette, avoid generating bubbles, wipe the surface moisture of the electroporation cuvette and place it in the electroporation chamber for electroporation. The electroporation parameters are set as: voltage 1.8 kV, resistance 200 Ω, capacitance 25 μF, and the ideal electroporation time is about 5 ms.

[0093] Immediately add 1 mL of LB broth, pipette and mix well, then transfer to a 1.5 mL EP tube. Incubate at 37 °C with shaking at 200 rpm for 1 h. Centrifuge at 8000 rpm for 2 min at room temperature to collect all the bacteria, and spread them on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apra. Incubate the plate upside down at 37 °C overnight.

[0094] Pick the suspected positive single colony with a sterile pipette tip and transfer it to 50 μL of 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 product, and screen the correct protein SecA A112V mutant strain according to the sequencing results, that is, the 112th amino acid of the SecA protein is mutated from alanine to valine.

[0095] 5.3 Elimination of pTAM112 plasmid

[0096] Using the SecA A112V mutant strain as the object, refer to the method and related materials in 4.2 to obtain the strain that has successfully eliminated pTAM112, named EF1005.

[0097] 6 Elimination of pEcCas plasmid

[0098] Transfer the obtained strain EF1005 to 3 mL of LB broth containing 15% sucrose, incubate at 37 °C with shaking at 200 rpm for 13 h. Use an inoculation loop to pick up a small amount of the cultured bacterial solution and streak it on an LB plate containing 15% sucrose. Incubate the plate upside down at 37 °C overnight.

[0099] Pick a single colony with a sterile pipette tip and transfer it to 50 μL of LB broth for expansion culture. Take 1 μL of the bacterial solution as a template and perform PCR verification with primers cas-F and cas-R. If there is no amplification product, pEcCas is successfully eliminated, and this strain is M112.

[0100] 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.

[0101] Table 1 Primer information

[0102]

[0103] Table 2 Strain information

[0104]

[0105] Table 3 Plasmid information

[0106]

[0107] Table 4 PCR reaction system

[0108]

[0109] Table 5 PCR reaction procedure

[0110]

[0111] Example 2

[0112] This example provides a method for constructing a conjugation transfer recipient bacterium. The specific steps and parameters are as follows:

[0113] 1. Loss of the endogenous plasmid pEFER of Escherichia fergusonii

[0114] Using the sodium dodecyl sulfate (SDS) culture method, inoculate the bacteria into 5 mL of LB broth medium, culture at 37 °C with shaking at 200 rpm for 16 h. Take 50 μL and inoculate it into 5 mL of LB broth containing 0.05% SDS, culture at 37 °C with shaking at 200 rpm for 16 h, and repeatedly passage in LB broth and LB broth containing SDS. When culturing to the 8th - 9th generation, streak the bacterial solution on a plate, select single colonies, amplify with plasmid-specific primers KP-F and KP-R, and detect the amplification result by electrophoresis. No band indicates obtaining the strain EF1001 with the loss of the endogenous plasmid pEFER of Escherichia fergusonii.

[0115] 2. Construction of the editing plasmid

[0116] 2.1 bla TEM-1B Construction of the knockout plasmid pTAKB

[0117] Using the wild-type Escherichia fergusonii genome as a template, amplify the upstream and downstream homologous arms HA1 and HA2 using primers bla-up-F (the homologous arm sequences contained in the primer are the thick underlined parts in SEQ ID NO.3 in Table 1), bla-up-R and bla-down-F (the homologous arm sequences contained in the primer are the thick underlined parts in SEQ ID NO.5 in Table 1), bla-down-R respectively bla TEM-1B ;

[0118] Using the pTA plasmid as a template, the plasmid backbone fragment Vbla-1 was amplified using primers V1-F and bla20-R (the gRNA sequences contained in the primers are the thick underlined parts in SEQ ID NO.10 in Table 1), and the plasmid backbone fragment Vbla-2 was amplified using primers bla20-F (the gRNA sequences contained in the primers are the thick underlined parts in SEQ ID NO.8 in Table 1) and V1-R;

[0119] The above PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5;

[0120] The above four obtained fragments were recovered and their concentrations were measured, and they were ligated by the method of DNA assembly technology (Gibson assembly). The ligation product was transformed into competent Escherichia coli DH10B cells and cultured on an LB plate containing 50 μg / mL apramycin sulfate (Apra) at 37 °C for 15 h. Subsequently, plasmid extraction, restriction enzyme digestion verification and sequencing alignment were carried out through a kit to obtain the correctly constructed recombinant plasmid pTAKB. The schematic diagram of the recombinant plasmid pTAKB is shown in Figure 1 .

[0121] 2.2 Construction of SecA (A112V) mutant plasmid pTAM112

[0122] Using the wild-type Escherichia fergusonii genome as a template, primers secA -up1-F / R and secA -up2-F / R were used to amplify the upstream homologous arm fragments HA3-1 and HA3-2 of the secA mutation site; Using HA3-1 and HA3-2 as templates, primers secA -up1-F and secA -up2-R were used to obtain the fusion fragment HA3 by PCR; Primers secA -down-F / R were used to amplify the downstream homologous arm fragment HA4 of the secA mutation site; Using the pTA plasmid as a template, primers V1-F and secA 20-R (the gRNA sequences contained in the primers are the thick underlined parts of SEQ ID NO.18 in Table 1) were used to amplify the plasmid backbone fragment V112-1; Primers secA 20-F (the gRNA sequences contained in the primers are the thick underlined parts of SEQ ID NO.17 in Table 1) and V1-R were used to amplify the plasmid backbone fragment V112-2;

[0123] The above PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5;

[0124] Determine the concentrations of fragments HA3, HA4, V112-1, and V112-2, ligate them by the method of Gibson assembly, transform the ligation product into Escherichia coli DH10B competent cells, and culture them on an LB plate containing 50 μg / mL Apra at 37 °C for 15 h. Subsequently, extract the plasmid using a kit, verify it by restriction digestion, and perform sequencing alignment to obtain the correctly constructed recombinant plasmid pTAM112. The map is as shown in Figure 2 shown.

[0125] 3. Introduction of gene editing plasmid

[0126] 3.1 Preparation of EF1001 competent cells

[0127] Use an inoculation loop to pick the strain EF1001 (glycerol-preserved bacteria at -80 °C), streak it on an LB medium without resistance, and culture it overnight at 37 °C in a constant temperature incubator. Pick a single colony from the above plate and inoculate it into 5 mL of LB broth without antibiotics. Culture it at 37 °C and 200 rpm for 14 h.

[0128] Inoculate the cultured bacterial solution at a ratio of 1:100 into 100 mL of LB liquid medium, culture it at 37 °C and 200 rpm. When the OD 600 value reaches 0.6 - 0.8 (about 2 h of culture), stop the culture. Aliquot the cultured bacterial solution into 50 mL centrifuge tubes, place it on ice for 10 min to stop growth. Place the centrifuge tubes in a pre-cooled centrifuge (4 °C), centrifuge at 4000 rpm and 4 °C for 10 min, and discard the supernatant. Add 15 mL of pre-cooled ddH 2 O to resuspend the cells, centrifuge at 4000 rpm and 4 °C for 10 min, and discard the supernatant. Add 15 mL of pre-cooled 10% glycerol to each centrifuge tube to resuspend the cells, centrifuge at 4000 rpm and 4 °C for 10 min, and discard the supernatant. Repeat this step once. Resuspend the cells with 500 μL of pre-cooled 10% glycerol, aliquot the EF1001 competent cells into 1.5 mL EP tubes, 40 μL per tube, and quickly store them at -80 °C after completion.

[0129] 3.2 Transformation of pEcCas plasmid into strain EF1001

[0130] Take out the EF1001 competent cells from the -80 °C refrigerator, place them on ice to melt, add 600 ng of pEcCas plasmid to 40 μL of the competent cells, and gently mix with a pipette tip.

[0131] Transfer the mixed solution into a clean and pre-cooled 1 mm electroporation cuvette, avoiding the generation of air bubbles. Wipe the surface moisture of the electroporation cuvette and place it in the electroporation chamber 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 of LB broth, pipette and mix well, and transfer it to a 1.5 mL EP tube. Incubate at 37 °C and 200 rpm for 1 h. Centrifuge at 8000 rpm at room temperature for 2 min to collect all the bacteria, and spread them on an LB plate containing 50 μg / mL kanamycin sulfate (Kan). Incubate the plate upside down at 37 °C overnight.

[0132] Pick the suspected positive single colony with a sterile pipette tip and transfer it to 3 mL of LB broth containing 40 μg / mL Kan for expanded culture. Take 1 μL of the bacterial solution as a template and perform PCR with the pEcCas specific primers cas-F / R. Name the correctly identified positive colony EF1002.

[0133] 3.3. Preparation of competent cells of strain EF1002

[0134] Pick strain EF1002 (glycerol stock stored at -80 °C) with an inoculation loop and streak it on an LB solid medium plate containing 50 μg / mL Kan antibiotic. Incubate it overnight at 37 °C in a constant temperature incubator. Pick a single colony from the above plate and inoculate it into 5 mL of LB broth containing 50 μg / mL Kan. Incubate at 37 °C and 200 rpm for 13 h.

[0135] Inoculate the cultured bacterial solution into 100 mL of LB liquid medium containing 50 μg / mL Kan and 30 mM L-arabinose at a volume ratio of 1:100. Incubate at 37 °C and 200 rpm. When the OD 600 value reaches 0.6 - 0.8 (about 2 h of culture), stop the culture. Aliquot the cultured bacterial solution into 50 mL centrifuge tubes and place them on ice for 10 min to stop growth.

[0136] Place the centrifuge tubes in a pre-cooled centrifuge (4 °C), centrifuge at 4000 rpm at 4 °C for 10 min, and discard the supernatant.

[0137] Add 15 mL of pre-cooled ddH 2 O to each centrifuge tube to resuspend the bacteria. Centrifuge at 4000 rpm at 4 °C for 10 min and discard the supernatant.

[0138] Add 15 mL of pre-cooled 10% glycerol to each centrifuge tube to resuspend the bacteria. Centrifuge at 4000 rpm at 4 °C for 10 min and discard the supernatant. Repeat this step once more.

[0139] Resuspend the cells with 500 μL of pre-cooled 10% glycerol, aliquot the competent cells into 1.5 mL EP tubes, 40 μL per tube, and quickly store at -80 °C after completion.

[0140] 4. secA Point mutation at amino acid position 112 of the gene

[0141] 4.1. Transformation of pTAM112 into EF1002 and mutation verification

[0142] Take out the EF1002 competent cells from the -80 °C refrigerator, place them on ice to melt, add 500 ng of pTAKB to 40 μL of competent cells, and gently mix with a pipette tip.

[0143] Transfer the mixture into a clean and pre-cooled 1 mm electroporation cuvette, avoiding the generation of bubbles. Wipe the surface moisture of the electroporation cuvette and place it in the electroporation chamber 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 of LB broth, mix well by pipetting and transfer to a 1.5 mL EP tube, and culture at 37 °C and 200 rpm for 1 h. Centrifuge at 8000 rpm at room temperature for 2 min to collect all the cells, and spread them on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apra, and incubate inverted at 37 °C overnight.

[0144] Pick the suspected positive single colonies with a sterile pipette tip into 50 μL of 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, perform PCR with primers M112-F and M112-R, sequence the sequencing products, and screen the correct SecA A112V mutant strain according to the sequencing results, that is, the 112th amino acid of the SecA protein is mutated from alanine to valine.

[0145] 4.2. Elimination of pTAM112 plasmid

[0146] Using the SecA A112V mutant strain as the target, refer to the method and related materials in 4.2 of Example 1 to obtain the strain that has successfully eliminated pTAM112, named EF1004.

[0147] 5. Transformation of pTAKB plasmid into strain EF1002 and knockout verification

[0148] 5.1. Transformation of pTAKB plasmid into the strain

[0149] Using the strain EF1004 as the basic cell, prepare EF1004 competent cells according to the method in 3.3.

[0150] Take out the EF1002 competent cells from the -80 °C refrigerator, place them on ice to melt, add 400 ng of pTAKB to 40 μL of competent cells, and gently mix with a pipette tip. Transfer the mixture into a clean and pre-cooled 1 mm electroporation cuvette, avoiding the generation of bubbles. Wipe the surface moisture of the electroporation cuvette and place it in the electroporation chamber 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 of LB broth, mix well by pipetting and blowing, and transfer it to a 1.5 mL EP tube. Incubate at 37 °C and 200 rpm for 1 h. Centrifuge at 8000 rpm at room temperature for 2 min to collect all the bacteria, and spread them on an LB plate containing 50 μg / mL Kan and 50 μg / mL Apra. Incubate the plate upside down at 37 °C overnight.

[0151] Pick the suspected positive single colonies with a sterile pipette tip and transfer them to 50 μL of 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 verification with primers blaK-F and blaK-R (the PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5). Screen according to the product size bla TEM-1B knockout strains.

[0152] 5.2 Elimination of plasmid pTAKB

[0153] Transfer the above-screened bla TEM-1B knockout strains to 3 mL of LB liquid medium containing 50 μg / mL Kan and 30 mM rhamnose, incubate at 37 °C and 200 rpm for 12 h. Use an inoculation loop to pick up a small amount of the cultured bacterial solution and streak it on an LB plate containing 50 μg / mL Kan. Incubate the plate upside down at 37 °C overnight.

[0154] Pick single colonies with a sterile pipette tip and transfer them to 50 μL of LB broth containing 50 μg / mL Kan for expansion culture. Take 1 μL of the bacterial solution as a template and perform PCR verification with primers TA-F and TA-R (the PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5). The absence of amplification products indicates the successful elimination of pTAKB, and the strain with correct identification is named EF1005.

[0155] 6 Elimination of pEcCas plasmid

[0156] The obtained strain EF1005 was transferred to 3 mL of LB broth containing 10% sucrose, cultured at 37 °C with 200 rpm for 13 h. A small amount of the cultured bacterial liquid was taken with an inoculation loop and streaked on an LB plate containing 10% sucrose, and cultured overnight at 37 °C in an inverted position.

[0157] A single colony was picked with a sterile pipette tip and transferred to 50 μL of LB broth for enlarged culture. 1 μL of the bacterial liquid was taken as a template, and PCR verification was carried out using primers cas-F and cas-R. If there was no amplification product, pEcCas was successfully eliminated, and this strain was M112.

[0158] 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.

[0159] Experimental Example 1

[0160] The strain M112 prepared in Example 1 of the present invention was cultured in LB broth containing different concentrations of sodium azide respectively. 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 in sequence.

[0161] Escherichia coli J53 was cultured in LB broth containing different concentrations of sodium azide respectively. 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 in sequence.

[0162] The culture conditions for each experimental group and control group were 37 °C, and the culture time was 24 h.

[0163] According to Figure 3 It can be seen that the strain M112 provided by the present invention has resistance to sodium azide.

[0164] Experimental Example 2

[0165] Citrobacter freundii ( Citrobacter freundii BC73) and Escherichia coli ECCRA-119 were used as conjugation transfer donor bacteria respectively, and the strain M112 obtained in Example 1 of the present invention and Escherichia coli J53 were used as conjugation transfer recipient bacteria. The conjugation transfer donor bacteria Citrobacter freundii BC73 carried bla NDM-1 gene, which was located on the IncFIB plasmid, and the conjugation transfer donor bacteria ECCRA-119 carried bla NDM-5 gene, which was located on the IncX3 plasmid.

[0166] Mix the donor bacteria and recipient bacteria in the logarithmic growth phase at a volume ratio of 1:1, and conjugate for 8 h at 37 °C in a non-resistant LB medium.

[0167] Collect the cells from the mixed culture, and perform gradient dilution spotting on an LB plate containing sodium azide (100 μg / mL) (the recipient bacteria J53 and strain M112 can grow), and a plate containing sodium azide (100 μg / mL) and meropenem (4 μg / mL) (the recipient bacteria that have successfully obtained the plasmid, i.e., the transconjugants, can grow). The conjugation transfer efficiency is the number of transconjugants / number of recipient bacteria.

[0168] The conjugation transfer results with Citrobacter freundii ( Citrobacter freundii BC73) as the donor bacteria are as shown in Figure 4 and Figure 5 The conjugation transfer results with Escherichia coli ECCRA-119 as the donor bacteria are as shown in Figure 6 and Figure 7 ( Figure 6 and Figure 7 The 10 -1 ~10 -6 indicates the dilution factor of the bacterial solution after conjugation transfer. Specifically, after conjugation transfer, the cells are collected and resuspended in 1 mL of normal saline to form a bacterial solution, which is the original bacterial solution. Dilute the original bacterial solution 10-fold to 10 6 times to represent 10 -1 ~10 -6 ). The conjugation transfer efficiency of the conjugation transfer recipient bacteria prepared in the examples of the present invention and Escherichia coli J53 is of the same order of magnitude, and the two have similar conjugation transfer efficiencies.

[0169] Obviously, the above examples are only for clear illustration and not a limitation of the implementation mode. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all implementation modes here. And the obvious changes or variations derived therefrom are still within the protection scope of the present 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. A method for constructing a conjugative transfer recipient bacterium, characterized in that: The steps include: Eliminating the endogenous plasmid of Escherichia Fergusonii, wherein the deposit number of the Escherichia Fergusonii is ATCC 35469, and the endogenous plasmid is plasmid pEFER; Introducing a gene editing plasmid into Escherichia fergusonii, wherein the gene editing plasmid is a pEcCas plasmid; Construct knockout bla TEM-1B The plasmid of the gene, the construction of 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 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 as shown in SEQ ID NO: 8 and SEQ ID NO: 9; Constructing a mutant plasmid in which the amino acid at position 112 of protein SecA is mutated from alanine to valine. The method for constructing a mutant plasmid in which the amino acid at position 112 of protein SecA is mutated from alanine to valine comprises amplifying the mutant 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 was amplified using Escherichia fergusonii as a template secA The downstream homology arm fragment of the mutation site was amplified using pTA plasmid as a template. secA The first plasmid backbone fragment of the mutation site was amplified using the pTA plasmid as a template. secA The second plasmid backbone fragment at the mutation site 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 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; 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.

3. The construction method according to claim 2, 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.

4. The construction method according to claim 2, 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.

5. The construction method according to claim 2, 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: 21 to SEQ ID NO: 22; The construction method also includes a step of eliminating the gene editing plasmid, wherein the step of eliminating the gene editing plasmid includes shaking culturing the strain introduced with the gene editing plasmid in a medium containing 10-20wt% sucrose for 12-14 hours, and streaking culturing in a medium containing 10-20wt% sucrose for at least 12 hours, and the verification primers for eliminating pEcCas are shown in SEQ ID NO: 25 to SEQ ID NO:

26.

6. Any of the following uses of the conjugation transfer recipient bacterium according to claim 1 or the conjugation transfer recipient bacterium constructed by the construction method of the conjugation transfer recipient bacterium according to any one of claims 2 to 5: (I) Detect the conjugative transfer efficiency of the donor bacterial plasmid; (ii) Engineered bacteria used for bacterial genetic manipulation.

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