A high-efficiency method for cultivating bovine gallstone strain marker

Abstract

A highly efficient labeling method for cultivating *Bezoar* strains, belonging to the field of *Bezoar* strain labeling, includes the following steps: Step 1: Preparation of a recombinant single fragment; Step 2: First-round fusion PCR; Step 3: Second-round fusion PCR; Step 4: Third-round fusion PCR, preparing the recombinant fragment lacZ ins EGFP-Kan-HR, precipitating and recovering with ethanol, and quantifying at 200 ng / μL; Step 5: Preparation of XD28 electroporation competent cells; Step 6: Transforming the RED recombinase system pKD46 into XD28 competent cells and inducing recombinant system expression; Step 7: Transforming the recombinant fragment; Step 8: Verifying the knock-in of the marker gene; Step 9: Verifying the function of the lacZ gene; Step 10: Passaging to remove the pKD46 plasmid; Step 11: Strain preservation. This invention enables stable expression of green fluorescent protein in this highly efficient *Bezoar*-cultivating strain, achieving long-term stable labeling.

Description

Technical Field

[0001] This invention belongs to the field of gene engineering and is specifically a marker method for cultivating Botrytis cinerea strains. Background Technology

[0002] XD28 is a microorganism proven to be highly effective in cultivating bezoar. Because it can efficiently induce bezoar production in vitro and in bovine gallbladder, precise genetic manipulation of it, especially reliable gene markers, is crucial for tracking its behavior in complex environments.

[0003] However, genetic modification of this yellow-producing strain faces significant challenges. Traditional gene marker methods, such as transposon insertion mutations or gene knockout / knock-in based on restriction endonucleases and ligases, are inefficient and cumbersome in this strain. This is mainly due to the large bacterial genome and extremely low natural transformation efficiency. Therefore, a marker method is urgently needed to facilitate timely behavioral tracking and localization of this strain.

[0004] Red recombination is a powerful tool that utilizes phage-encoded Exo, Beta, and Gam proteins to mediate efficient recombination of linear double-stranded DNA fragments with chromosomal target sites via short homologous arms (typically 35-50 bp) (cited in: One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products). This technology has demonstrated significant advantages in model bacteria such as Escherichia coli, including high efficiency, speed, and site specificity (cited in: Antibiotic marker modifications of lambdaRed and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains; Application of lambdaRed recombination system to Vibrio cholerae genetics: simple methods for inactivation and modification of chromosomal genes; Structure and mechanism of the Red recombination system of bacteriophage λ).

[0005] Despite the immense potential of Red recombination technology, its direct application to gene markers in strains that efficiently cultivate bezoar has not yet been successfully reported. Introducing this system into non-model strains often faces numerous obstacles, including difficulties in heterologous expression of the Red operon and the need to optimize recombination conditions (such as electroporation parameters and induction timing) for specific strains. Therefore, developing a high-efficiency gene marker platform technology based on the Red recombination principle, adapted to the highly efficient bezoar-cultivating strain XD28, to overcome the bottlenecks of existing genetic manipulation methods has become an urgent need to promote related research and applications of this strain. Summary of the Invention

[0006] This invention provides a labeling method for the efficient cultivation of Bezoar strains, thereby overcoming the deficiencies in the prior art.

[0007] This invention is achieved through the following technical solution:

[0008] A labeling method for efficiently cultivating Bezoar strains includes the following steps:

[0009] Step 1: Synthesize the J23119(SpeI) promoter, a constitutive promoter used to initiate the expression of downstream genes. The sequence is TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGT. An insertion site was designed at the C-terminus of the lacZ gene, with an upstream homologous arm 5HR and a downstream homologous arm 3HR. Using the E. coli XD28 genome as a template, the 5HR and 3HR homologous arms were amplified, with expected sizes of 206 bp and 235 bp, respectively. The PCR amplification products were subjected to 1.0% agarose gel electrophoresis. The resulting single band was excised and recovered to obtain purified upstream and downstream homologous arms 5HR and 3HR. Using plasmid pLV-EGFP-N containing the green fluorescent protein (EGFP) marker gene as a template, and plasmid pET28a containing the resistance selection gene KanR and the T7 terminator as a template, fragments EGFP, KanR, and T7 terminator were amplified to obtain purified EGFP, KanR, and T7 terminator fragments.

[0010] Step 2: Fuse the J23119 and 5HR fragments, EGFP-N, T7ter and 3HR fragments obtained in Step 1 into three fusion fragments, resulting in the J23119+5HR fragment, EGFP-N, T7ter+3HR fusion fragment;

[0011] Step 3: Using the three fusion fragments J23119+5HR, EGFP-N, and T7ter+3HR obtained in Step 2 as templates, perform fusion PCR reactions of 5HR-J23119 with EGFP-N and Kan with T7ter+3HR to obtain purified fusion fragments 5HR+J23119+EGFP-N and Kan+T7ter+3HR.

[0012] Step 4: Using the fusion fragments 5HR+J23119+EGFP-N and Kan+T7ter+3HR obtained in Step 3 as templates, a fusion PCR reaction of the fragments was performed. The PCR product was recovered by ethanol precipitation and quantified at 200 ng / μL to obtain the purified recombinant fragment lacZ ins EGFP-Kan-HR.

[0013] Step 5: Prepare electrocompetent cells of strain XD28. Resuscitate strain XD28 preserved in glycerol using the four-zone streak method. Incubate on LB agar plates at 37°C for 14-16 h. Pick single colonies from the plates and culture in 2 mL LB agar at 30°C until OD600 = 0.4-0.6. Transfer 1 mL to 1.5 mL from each single colony culture sample into centrifuge tubes. Cool the cells in an ice-water bath for 10-15 minutes. Centrifuge at 3000 rpm for 10 min at 4°C. Discard the supernatant using a pipette. Gently resuspend the cells in 1 mL of pre-chilled dH2O. Centrifuge at 3000 rpm for 10 min at 4°C. Gently resuspend the cells again in 500 μL of pre-chilled 10% glycerol. Repeat 3-5 times, centrifuging at 3000 rpm for 10 min at 4°C. Transfer the cells to 50 μL of pre-chilled 10% glycerol. The cells were gently resuspended in ice water;

[0014] Step Six: Transform the Red recombinase plasmid pkd46, containing Gam, Beta, and Exo from the Red recombinant system, into the XD28 electroporation competent cells prepared in Step Five using electroporation. Add 2 μg of Red recombinase plasmid pKD46 to 100 μL of competent cells, mix gently, and transfer to an electrode cup pre-chilled on ice. Set the electroporator parameters to 2.5 kV, 600 Ω, and perform electroporation according to the default pulse time of the electroporator. Immediately after electroporation, add 1 mL of antibiotic-free LB liquid medium. After recovery, spread all cells onto antibiotic plates and incubate at 30°C for 12-14 h. Pick clones grown in Amp antibiotic plates and transfer them to Amp liquid medium. Incubate at 30°C until OD=0.3, then add 0.2% arabinose to induce Red recombinase expression. Continue culturing until OD=0.5, and prepare electroporation competent cells XD28-pkd46 according to the method in Step Five.

[0015] Step 7: Transform 1 μL of the recombinant fragment lacZ ins EGFP-kan-HR into 50 μL of electrocompetent cells XD28-pkd46 using the electroporation method described in Step 6. Add 1 ml of antibiotic-free LB medium and incubate at 30°C for 2 h. Centrifuge the culture at 3000 rpm for 5 minutes, discard the supernatant, resuspend the culture in 100 μL of antibiotic-free LB liquid medium, and spread the entire culture evenly onto KanR LB solid culture plates containing 0.2% L-arabinose. Incubate at 30°C for 20 h and then check for the growth of colony clones.

[0016] Step 8: Verify the knock-in of the EGFP-Kan marker gene. Select the growing colonies and clone them into KanR liquid medium containing 0.2% L-arabinose. Incubate at 30℃ for 6-8 h. Using the cloned bacterial culture as a template and E. coli XD28 strain as a control, perform PCR reaction according to the reaction system in Table 18. Perform PCR product electrophoresis on 1.0% agarose gel to identify whether the amplified fragment of the positive clone bacterial culture has knocked in the EGFP-Kan marker gene.

[0017] Step 9: lacZ gene function verification. Select the first positive clone from the above screening and culture it until OD600=0.4. Divide the clone into two tubes. One tube is induced with 0.4mM IPTG, and the other tube is not induced with IPTG. Use E. coli XD28 and E. coli XD28-pKD46 strains as controls. After culturing at 37℃ for 12-14 h, streak the bacterial culture onto LB agar plates containing X-gal. Compare the blue-white colonies of E. coli XD28, E. coli XD28-pKD46, and lacZ ins EGFP-kan-XD28 clone 1. If all three show white or light blue colonies without induction and dark blue colonies after IPTG induction, it indicates that the lacZ gene at the knock-in site is functioning normally.

[0018] Step 10: Removal of pKD46 plasmid from the Red recombinase system. The lacZinsEGFP-kan-XD28 clone 1 was passaged at 37℃ to remove the pKD46 plasmid. After passage, the bacterial culture was plated on AmpR-resistant LB solid medium. If it no longer grows on AmpR LB solid medium, it proves that the plasmid removal was successful. The bacterial culture after plasmid removal was then purified by streak plating in four zones. The purified bacterial culture with the EGFP-Kan marker gene successfully knocked in was named XD28E.

[0019] Step 11: Culture preservation. Select purified XD28E single clones for overnight culture. Mix the culture medium with 75% glycerol in a 1:1 ratio and preserve the culture at -80℃.

[0020]

[0021] The labeling method for efficiently cultivating Bezoar strains as described above, wherein the EGFP sequence is:

[0022] The labeling method for efficiently cultivating Bezoar strains as described above, wherein the LB plate of Kan in step three contains 0.2% L-arabinose.

[0023] The labeling method for efficient cultivation of Bezoar strains described above, in step six, the conclusion of lacZ gene function verification is as follows: without IPTG induction, all three types of colonies are white or light blue; with IPTG induction, all three types of colonies are dark blue; the experimental results of the three types are consistent, and the mutation does not affect the function of the lacZ gene.

[0024] The advantages of this invention are: This invention knocks in the EGFP-kan gene, a marker gene for the strain, into the genome of the high-efficiency bezoar-producing strain XD28 obtained through screening. The knock-in location is at the C-terminus of the lacZ gene, without disrupting the expression of the lacZ gene, allowing green fluorescent protein to be stably expressed in this high-efficiency bezoar-producing strain, thus achieving long-term stable labeling. This invention selects the C-terminus of the lacZ gene as the insertion site for the EGFP-kan marker gene. After knocking in the EGFP-kan marker gene, the expression of the lacZ gene is not disrupted, and the bacteria can grow normally in the culture medium without plasmid residue and possess the same biological characteristics as the pre-labeled bezoar-producing strain XD28. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the construction and insertion site of the recombinant fragment according to an embodiment of the present invention;

[0027] Figure 2 This is a schematic diagram of the PCR identification results of knock-in positive monoclonal bacterial culture in an embodiment of the present invention;

[0028] Figure 3 This is one of the schematic diagrams showing the functional verification results of the lacZ gene in an embodiment of the present invention;

[0029] Figure 4 This is the second schematic diagram of the lacZ gene function verification results in an embodiment of the present invention;

[0030] Figure 5 This is a fluorescence observation diagram of an embodiment of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] A labeling method for efficiently cultivating Bezoar strains includes the following steps:

[0033] Step 1: Synthesize the J23119(SpeI) promoter, a constitutive promoter used to initiate the expression of downstream genes. The sequence is TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGT. An insertion site was designed at the C-terminus of the lacZ gene, with an upstream homologous arm 5HR and a downstream homologous arm 3HR. Using the E. coli XD28 genome as a template, the 5HR and 3HR homologous arms were amplified, with expected sizes of 206 bp and 235 bp, respectively. The PCR amplification products were subjected to 1.0% agarose gel electrophoresis. The resulting single band was excised and recovered to obtain purified upstream and downstream homologous arms 5HR and 3HR. Using plasmid pLV-EGFP-N containing the green fluorescent protein (EGFP) marker gene as a template, and plasmid pET28a containing the resistance selection gene KanR and the T7 terminator as a template, fragments EGFP, KanR, and T7 terminator were amplified to obtain purified EGFP, KanR, and T7 terminator fragments.

[0034] Step 2: Fuse the J23119 and 5HR fragments, EGFP-N, T7ter and 3HR fragments obtained in Step 1 into three fusion fragments, resulting in the J23119+5HR fragment, EGFP-N, T7ter+3HR fusion fragment;

[0035] Step 3: Using the three fusion fragments J23119+5HR, EGFP-N, and T7ter+3HR obtained in Step 2 as templates, perform fusion PCR reactions of 5HR-J23119 with EGFP-N and Kan with T7ter+3HR to obtain purified fusion fragments 5HR+J23119+EGFP-N and Kan+T7ter+3HR.

[0036] Step 4: Using the fusion fragments 5HR+J23119+EGFP-N and Kan+T7ter+3HR obtained in Step 3 as templates, a fusion PCR reaction of the fragments was performed. The PCR product was recovered by ethanol precipitation and quantified at 200 ng / μL to obtain the purified recombinant fragment lacZ ins EGFP-Kan-HR.

[0037] Step 5: Prepare electrocompetent cells of strain XD28. Resuscitate strain XD28 preserved in glycerol using the four-zone streak method. Incubate on LB agar plates at 37°C for 14-16 h. Pick single colonies from the plates and culture in 2 mL LB agar at 30°C until OD600 = 0.4-0.6. Transfer 1 mL to 1.5 mL from each single colony culture sample into centrifuge tubes. Cool the cells in an ice-water bath for 10-15 minutes. Centrifuge at 3000 rpm for 10 min at 4°C. Discard the supernatant using a pipette. Gently resuspend the cells in 1 mL of pre-chilled dH2O. Centrifuge at 3000 rpm for 10 min at 4°C. Gently resuspend the cells again in 500 μL of pre-chilled 10% glycerol. Repeat 3-5 times, centrifuging at 3000 rpm for 10 min at 4°C. Transfer the cells to 50 μL of pre-chilled 10% glycerol. The cells were gently resuspended in ice water;

[0038] Step Six: Transform the Red recombinase plasmid pkd46, containing Gam, Beta, and Exo from the Red recombinant system, into the XD28 electroporation competent cells prepared in Step Five using electroporation. Add 2 μg of Red recombinase plasmid pKD46 to 100 μL of competent cells, mix gently, and transfer to an electrode cup pre-chilled on ice. Set the electroporator parameters to 2.5 kV, 600 Ω, and perform electroporation according to the default pulse time of the electroporator. Immediately after electroporation, add 1 mL of antibiotic-free LB liquid medium. After recovery, spread all cells onto antibiotic plates and incubate at 30°C for 12-14 h. Pick clones grown in Amp antibiotic plates and transfer them to Amp liquid medium. Incubate at 30°C until OD=0.3, then add 0.2% arabinose to induce Red recombinase expression. Continue culturing until OD=0.5, and prepare electroporation competent cells XD28-pkd46 according to the method in Step Five.

[0039] Step 7: Transform 1 μL of the recombinant fragment lacZ ins EGFP-kan-HR into 50 μL of electrocompetent cells XD28-pkd46 using the electroporation method described in Step 6. Add 1 ml of antibiotic-free LB medium and incubate at 30°C for 2 h. Centrifuge the culture at 3000 rpm for 5 minutes, discard the supernatant, resuspend the culture in 100 μL of antibiotic-free LB liquid medium, and spread the entire culture evenly onto KanR LB solid culture plates containing 0.2% L-arabinose. Incubate at 30°C for 20 h and then check for the growth of colony clones.

[0040] Step 8: Verify the knock-in of the EGFP-Kan marker gene. Select the growing colonies and clone them into KanR liquid medium containing 0.2% L-arabinose. Incubate at 30℃ for 6-8 h. Using the cloned bacterial culture as a template and E. coli XD28 strain as a control, perform PCR reaction according to the reaction system in Table 18. Perform PCR product electrophoresis on 1.0% agarose gel to identify whether the amplified fragment of the positive clone bacterial culture has knocked in the EGFP-Kan marker gene.

[0041] Step 9: lacZ gene function verification. Select the first positive clone from the above screening and culture it until OD600=0.4. Divide the clone into two tubes. One tube is induced with 0.4mM IPTG, and the other tube is not induced with IPTG. Use E. coli XD28 and E. coli XD28-pKD46 strains as controls. After culturing at 37℃ for 12-14 h, streak the bacterial culture onto LB agar plates containing X-gal. Compare the blue-white colonies of E. coli XD28, E. coli XD28-pKD46, and lacZ ins EGFP-kan-XD28 clone 1. If all three show white or light blue colonies without induction and dark blue colonies after IPTG induction, it indicates that the lacZ gene at the knock-in site is functioning normally.

[0042] Step 10: Removal of pKD46 plasmid from the Red recombinase system. The lacZinsEGFP-kan-XD28 clone 1 was passaged at 37℃ to remove the pKD46 plasmid. After passage, the bacterial culture was plated on AmpR-resistant LB solid medium. If it no longer grows on AmpR LB solid medium, it proves that the plasmid removal was successful. The bacterial culture after plasmid removal was then purified by streak plating in four zones. The purified bacterial culture with the EGFP-Kan marker gene successfully knocked in was named XD28E.

[0043] Step 11: Culture preservation. Select purified XD28E single clones for overnight culture. Mix the culture medium with 75% glycerol in a 1:1 ratio and preserve the culture at -80℃.

[0044]

[0045] Specifically, the EGFP sequence described in this embodiment is as follows:

[0046] Furthermore, in step three of this embodiment, the LB tablets containing kan contain 0.2% L-arabinose.

[0047] Furthermore, the conclusion of the lacZ gene function verification in step six of this embodiment is as follows: without IPTG induction, all three colonies are white or light blue; with IPTG induction, all three colonies are dark blue; the experimental results of the three are consistent, and the mutation does not affect the function of the lacZ gene.

[0048] Example

[0049] 1. Preparation of competent cells of host strain XD28

[0050] 1.1 Preparation of electrocompetent cells

[0051] The strain XD28, preserved in glycerol, was revived using the four-zone streak method and incubated on LB solid culture plates at 37°C for 14-16 hours.

[0052] Select a single clone from the plate and culture it in 2 mL LB at 30°C until OD600 = 0.4-0.6. Take 1 mL from each single clone culture sample and transfer it to a 1.5 mL centrifuge tube.

[0053] Cool the cells in an ice-water bath for 10-15 minutes, then centrifuge at 3000 rpm for 10 minutes at 4°C.

[0054] Use a pipette to aspirate / discard the supernatant and gently resuspend the cells in 1 mL of pre-chilled dH2O.

[0055] Centrifuge at 3000 rpm for 10 min at 4°C, then gently resuspend the cells in 500 μL of pre-cooled 10% glycerol. Repeat 3-5 times.

[0056] Centrifuge at 3000 rpm for 10 min at 4°C, and gently resuspend the cells in 50 μL of ice water.

[0057] 2. Red recombination system and preparation and transformation of recombination template

[0058] 2.1 Preparation of Recombinant Templates

[0059] pKD46 contains three enzymes of the Red homologous recombination system: Gam, Beta, and Exo.

[0060] 2.2 Preparation of recombinant lacZinsEGFP-kan-HR fragment

[0061] Recombinant arms 5HR and 3HR were designed at the C-terminus of the lacZ gene. The recombinant fragment lacZinsEGFP-kan-HR was amplified by PCR, recovered by ethanol precipitation, and quantified at a concentration of 200 ng / µl or higher.

[0062] 2.2.1 Primer Design

[0063] Table 1. Preparation process and primer design of recombinant lacZins EGFP-kan-HR fragment

[0064]

[0065] Preparation of recombinant fragments

[0066] The upstream homologous arm 5HR and the downstream homologous arm 3HR of the insertion site were designed. Using the E. coli XD28 genome as a template, the 5HR and 3HR homologous arms were amplified according to the systems in Tables 2 and 3 and the procedure in Table 7, with expected sizes of 206 bp and 235 bp, respectively. The PCR amplification products were subjected to 1.0% agarose gel electrophoresis, and the obtained single bands were excised and recovered to obtain the purified upstream and downstream homologous arms 5HR and 3HR, which were stored at -20℃ for later use.

[0067] Table 2. 5HRPCR reaction system for the upstream homologous arm of the insertion site.

[0068]

[0069] Table 3. 3HRPCR reaction system of the downstream homologous arm of the insertion site

[0070]

[0071] Using plasmid pLV-EGFP-N containing the green fluorescent protein (EGFP) marker gene as a template, and plasmid pET28a containing the resistance selection genes KanR and T7terminator as a template, fragments EGFP, KanR, and T7terminator were amplified according to the procedure in Table 7. The expected sizes were 736 bp, 812 bp, and 142 bp, respectively. The PCR amplification products were subjected to 1.0% agarose gel electrophoresis, and the obtained single bands were excised and recovered to obtain purified EGFP, KanR, and T7terminator fragments, which were stored at -20℃ for later use.

[0072] Table 4. EGFP-PCR reaction system for green fluorescent protein marker genes.

[0073]

[0074] Table 5. PCR reaction system for resistance selection gene KanR

[0075]

[0076] Table 6. PCR reaction system for the T7 terminator transcription terminator

[0077]

[0078] Table 7. PCR reaction procedure for single-fragment amplification of recombinant fragments.

[0079]

[0080] Number of cycles: From Step 2 to Step 4 35 cycles

[0081] The J23119 constitutive promoter was synthesized according to the reaction system in Table 8 and the reaction procedure in Table 9, in preparation for subsequent fragment fusion.

[0082] Table 8. PCR reaction system for promoter J23119 synthesis

[0083]

[0084] Table 9. PCR reaction procedure for promoter J23119 synthesis

[0085]

[0086] Number of cycles: From Step 2 to Step 4 35 cycles

[0087] A second round of PCR was performed, fusing the previously prepared J23119 and 5HR fragments, EGFP-N, T7ter, and 3HR fragments into three fusion fragments. The fusion reactions were carried out according to the reaction systems in Tables 10, 11, and 12, and the reaction procedures in Table 13. The resulting PCR products were subjected to 1.0% agarose gel electrophoresis, with expected sizes of 272 bp, 761 bp, and 356 bp, respectively. The individual fragments were then excised and recovered from the gel to obtain purified J23119+5HR, EGFP-N, and T7ter+3HR fusion fragments.

[0088] Table 1. PCR reaction system for 05HR+J23119 fragment fusion

[0089]

[0090] Table 11 EGFP-N fragment PCR reaction system

[0091]

[0092] Table 12 T7ter+3HR fragment fusion PCR reaction system

[0093]

[0094] Table 13 Fragment Fusion PCR Reaction Procedure

[0095]

[0096] Number of cycles: 10 cycles from Step 2 to Step 4 and 15 cycles from Step 5 to Step 7

[0097] A third round of PCR was performed, using the fusion fragments obtained in the second round as templates. Following the PCR reaction systems in Tables 14 and 15 and the reaction procedure in Table 13, fusion PCR reactions were conducted on two fusion fragments: 5HR-J23119 with EGFP-N and Kan with T7ter+3HR. The resulting PCR products were subjected to 1.0% agarose gel electrophoresis, with expected sizes of 1013 bp and 1144 bp, respectively. The individual fragments were then excised and recovered from the gel to obtain purified fusion fragments 5HR+J23119+EGFP-N and Kan+T7ter+3HR.

[0098] Table 14 Fusion PCR reaction of 5HR-J23119+EGFP-N

[0099]

[0100] Table 15 Fusion PCR reaction of Kan+T7ter+3HR

[0101]

[0102] A fourth round of PCR was performed, using the previously obtained fusion fragments 5HR+J23119+EGFP-N and Kan+T7ter+3HR as templates. The PCR reaction was conducted according to the PCR reaction system in Table 16 and the PCR reaction procedure in Table 17, with four tubes used. The expected size was 2136 bp. The PCR product was recovered by ethanol precipitation, and the concentration was quantified at 200 ng / μL to obtain the purified recombinant fragment lacZinsEGFP-Kan-HR.

[0103] Table 16 PCR reaction system for the lacZinsEGFP-Kan-HR recombinant fragment

[0104]

[0105] Table 17 PCR reaction procedure for the lacZinsEGFP-Kan-HR recombinant fragment

[0106]

[0107] 2.3 Transformation of pKD46 plasmid

[0108] Cool the electrode cup on ice for at least 5 minutes. Prepare E. coli XD28 competent cells according to the method for preparing competent cells in 1.1. Add 2 μg of Red recombinase plasmid pKD46 to 100 μL of competent cells, mix gently, and transfer to the ice-cooled electrode cup. Set the electroporator parameters to 2.5 kV, 600 Ω, and perform electroporation transformation according to the default pulse time of the electroporator. Immediately after electroporation, add 1 mL of antibiotic-free LB liquid medium and incubate at 30°C and 180 rpm for 2-3 h to revive the competent cells. Centrifuge the culture medium at 3000 rpm for 5 minutes, discard the supernatant, resuspend the cells in 100 μL of antibiotic-free LB liquid medium, and spread the entire culture onto AmpRLB solid culture plates. After incubation for 18-20 h, check for the growth of colony clones. Select the grown clones and culture them at 30℃ until OD600=0.3. Add 0.2% L-arabinose to induce the expression of Red recombinase. Continue to culture at 30℃ until OD600=0.5. Then, prepare electroporation competent cells E.coliXD28-pKD46 according to the competent cell preparation method in 1.1.

[0109] 2.4 Transformation of the recombinant fragment lacZinsEGFP-Kan-HR

[0110] Transform 1 μL of the recombinant fragment lacZinsEGFP-Kan-HR into 100 μL of electroporation competent XD28-pKD46 cells using the electroporation method described in section 2.3. After electroporation, add 1 mL of antibiotic-free LB broth and incubate at 30°C and 180 rpm for 2–3 h to resuscitate the competent cells. Centrifuge the culture at 3000 rpm for 5 min, discard the supernatant, resuspend the cells in 100 μL of antibiotic-free LB broth, and evenly spread the entire culture onto KanRLB solid culture plates containing 0.2% L-arabinose. Incubate at 30°C for 20 h and then examine for colony growth.

[0111] 2.5 Validation of EGFP-Kan marker gene knock-in

[0112] Select the growing colonies and clone them into KanR liquid medium containing 0.2% L-arabinose. Incubate at 30°C for 6-8 hours. Using the cloned bacterial culture as a template and E. coli XD28 strain as a control, perform PCR reaction according to the reaction system in Table 18. Perform PCR product electrophoresis on 1.0% agarose gel to identify whether the amplified fragment of the positive cloned bacterial culture has knocked in the EGFP-Kan marker gene. If the knock-in is successful, the length of the amplified fragment should be 2134 bp.

[0113] Table 18 PCR Identification Reaction System for Positive Clones

[0114]

[0115] 2.6 Functional identification, purification, and plasmid removal of knock-in strains

[0116] 2.6.1 Functional verification of the lacZ gene

[0117] The first positive clone selected above was cultured until OD600 = 0.4, then divided into two tubes. One tube was induced with 0.4 mM IPTG, while the other tube was not induced with IPTG. E. coli XD28 and E. coli XD28-pKD46 strains were used as controls. After incubation at 37℃ for 12-14 h, the bacterial culture was streaked onto LB agar plates containing X-gal. The blue-white colony phenomenon of E. coli XD28, E. coli XD28-pKD46, and lacZinsEGFP-kan-XD28 clone 1 was compared. If all three colonies showed white or light blue colonies without induction and dark blue colonies after IPTG induction, it indicates that the lacZ gene at the knock-in site is functioning normally.

[0118] 2.6.2 Removal of pKD46 plasmid from the Red recombinase system and strain preservation

[0119] The first positive clone with normal verification function was taken and passaged at 37℃ to remove the pKD46 plasmid. After passage, the bacterial culture was plated on AmpR-resistant LB agar. If it no longer grew on AmpRLB agar, the plasmid removal was successful. The bacterial culture after plasmid removal was then purified by streak plating in four zones. The purified bacterial strain with the successfully knocked-in EGFP-Kan marker gene was named XD28E.

[0120] Select purified XD28E single clones for overnight culture, mix them with 75% glycerol at a ratio of 1:1, and store the strain at -80℃.

[0121] 3. Results

[0122] 3.1 Results of Recombinant Fragment Construction

[0123] After precipitation and fragment recovery, the fragment was sequenced, and lacZinsEGFP-Kan-HR was successfully constructed. The insertion site and elements are as follows. Figure 1 As shown.

[0124] 3.2 PCR identification results of positive monoclonal bacterial cultures after knock-in

[0125] Upon identification, clones 1-7 were all positive clones. The EGFP-Kan marker gene was successfully knocked into all clones at this locus compared to the XD28 strain genome. Lane descriptions from left to right: (wild-type 766bp / positive 2134bp) Lane 1: DL2000 DNA Marker; Lane 2: E. coli XD28; Lanes 3-9: lacZinsEGFP-kan-XD28 clones 1-7. Based on the identification results, clones 1-7 were all positive clones and were retained for future use. PCR sequencing of the genomes of clones 1-4 revealed that clone 1 had a non-synonymous mutation at 5HR (the mutation is located in the lacZ gene, e.g., ...). Figure 2 (As shown). Sequencing results are attached.

[0126] 3.3 Validation of lacZ gene function

[0127] To verify that the genomic point mutation of clone 1 does not affect lacZ function, a blue-white screening test was further performed. The results showed that without IPTG induction, all three colonies were white or pale blue (e.g., Figure 3 As shown); when IPTG was added for induction, all three colonies were dark blue (as shown). Figure 4 As shown in the figure); the experimental results of the three studies are consistent, indicating that the mutation does not affect the function of the lacZ gene.

[0128] 3.4 Verification of Fluorescence Display Effect

[0129] The fluorescence observation image of the labeled invention is shown below. Figure 5 As shown, the effect of fluorescent labeling is obvious and the fluorescent labeling is well completed.

[0130] It should also be noted that the XD28E strain is a self-selected strain and has been biologically preserved. The preservation center is the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 34799, the classification name is Escherichia coli, and the preservation date is July 7, 2025.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

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