A method for preparing a pulsed field gel electrophoresis marker of escherichia coli based on linear large fragment DNA

Abstract

The present application relates to a method for preparing a pulsed field gel electrophoresis marker based on Escherichia coli containing linear large fragment DNA. The present application prepares a pulsed field gel electrophoresis marker by selecting a plurality of Escherichia coli containing linear DNA of different lengths, mixing and digesting them quickly, and does not need to extract large fragment DNA additionally in the preparation process. According to the needs, the combination of Escherichia coli containing linear DNA of different lengths can be selected to customize the pulsed field gel electrophoresis marker containing specific bands and specific separation range. The pulsed field gel electrophoresis marker prepared by the method has the characteristics of uniform band distribution, customizable band size, simple and quick preparation process, and low preparation cost, and is suitable for molecular weight calibration and quality control of pulsed field gel electrophoresis experiments, and can be used for pulsed field gel electrophoresis analysis in the fields of microbial typing and genome mapping.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a method for preparing pulsed-field gel electrophoresis markers based on Escherichia coli containing large linear DNA fragments. Background Technology

[0002] Pulsed-field gel electrophoresis (PFGE) is an electrophoresis technique developed by Schwartz and Cantor in 1984. [1] Pulsed-field gel electrophoresis (PLGE) is a key technology for separating large DNA fragments. It achieves separation of high-molecular-weight DNA by periodically changing the direction of the electric field, effectively separating DNA fragments ranging from 10kb to 10Mb. It is an indispensable technique in fields such as microbial typing, drug-resistant bacteria analysis, infectious disease tracing, and foodborne disease surveillance. The accuracy of PLAGE results and the comparability of different batches depend on the DNA molecular weight standards (DNA markers) used. However, existing PLAGE DNA markers suffer from uneven band distribution, cannot be customized to specific band sizes, and are costly to manufacture.

[0003] Currently, commercially available PFGE DNA markers mainly include yeast chromosomal DNA markers and λ phage DNA tandem ladder markers. Yeast chromosomal DNA markers are produced by digesting yeast cells after cell wall removal. Their banding pattern is fixed at the size of a yeast chromosome, with some bands clustering unevenly, making it impossible to customize specific band sizes. The production process requires removing the yeast cell wall before digestion, typically taking 3-4 days. λ phage DNA tandem ladder markers are derived from the enzymatic digestion of recombinant λ phage DNA. This process requires the use of numerous specialized enzymes such as restriction enzymes and recombinases, resulting in high production costs and expensive market prices. *E. coli*, as a commonly used model organism, has a clear genetic background and is simple and inexpensive to cultivate. *E. coli* can serve as an excellent host for large-fragment DNA inheritance. Extracting BAC plasmids from *E. coli* and then digesting them in vitro to obtain DNA bands of different sizes can be used to produce PFGE DNA markers. r[2] However, this method requires the extraction of BAC plasmids followed by enzyme digestion, which is cumbersome and the cost of using restriction enzymes is high.

[0004] The references are as follows: [1]Schwartz, DC and CR Cantor, Separation of Yeast Chromosome- Sized DNAs by Pulsed Field Gradient Gel Electrophoresis. Cell, 1984. 37(1):p. 67-75. [2]Wong, YC, et al., Customizable BAC-based DNA markers for pulsed- field gel electrophoresis. Analytical Biochemistry, 2024. 693. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments. This invention rapidly prepares pulsed-field gel electrophoresis markers by mixing and digesting multiple *E. coli* strains containing linear DNA of different sizes. The preparation process does not require additional extraction of large DNA fragments, and different combinations of *E. coli* strains containing linear DNA of varying sizes can be selected to customize pulsed-field gel electrophoresis markers with specific bands and separation ranges as needed. Pulsed-field gel electrophoresis markers prepared using this method feature uniform band distribution, customizable band sizes, and a simple, rapid, and low-cost preparation process. They are suitable for molecular weight calibration and quality control in pulsed-field gel electrophoresis experiments and can be used for pulsed-field gel electrophoresis analysis in fields such as microbial typing and genome mapping.

[0006] The objective of this invention can be achieved through the following technical solutions: A method for preparing pulsed-field gel electrophoresis markers based on E. coli containing large linear DNA fragments, the specific steps of which are as follows: S1. Culture and mix various E. coli strains containing linear DNA to obtain mixed bacterial cells. Among them, the linear DNA contained in each type of Escherichia coli is of different length; S2. The mixed bacterial cells obtained in step S1 are embedded in a low-melting-point agarose gel to obtain a gel block; S3. Perform E. coli cell lysis and protein digestion on the gel block obtained in step S2 to obtain a gel block containing linear DNA bands of different lengths. S4. The gel block containing linear DNA bands of different lengths obtained in step S3 is separated by pulsed-field gel electrophoresis, which is the pulsed-field gel electrophoresis marker.

[0007] Furthermore, in step S1, the length of the linear DNA in the E. coli ranges from 10 Kb to 3000 Kb.

[0008] Further, in step S1, the mixed bacterial cells are selected from any two or more combinations of MT-pLW, MT-pLC-1, MT-pLC-2, MT-PZT, MT-pLC-3, p6-Liner, p1, pGXD5, pA, pB, pGXC5, pGXE3, pGXD8, pGXE4, ELC121-pWT1284, pGXG1, ELC121-90-1, ELC121-90-2, and pGXE6.

[0009] Further, in step S1, the E. coli containing linear DNA is obtained by the following method: A series of E. coli strains containing linear DNA were obtained by custom splicing through E. coli conjugation transfer and homologous recombination, and high-density cells of each strain were obtained through amplification culture.

[0010] As a preferred technical solution, the method for assembling linear-BAC iterative DNA in E. coli is referenced in the article Zhong, L., et al., The conjugation-associated linear-BAC iterative assembling (CALBIA) method for cloning 2.1-Mb human chromosomal DNAs in bacteria. . Cell Research,2025. 35(4): p. 309-312. Furthermore, in step S1, various types of *E. coli* containing linear DNA are subjected to overnight amplification culture to obtain bacterial cells, and the OD of each bacterial cell is measured. 600 OD of each strain 600 Mix the bacterial cells evenly in quantities of 0.1-1 to obtain mixed bacterial cells.

[0011] Further, in step S2, the low-melting-point agarose gel is prepared by mixing low-melting-point agarose powder with TE25S buffer and heating to melt, and the final concentration of the low-melting-point agarose powder is 1-2% (w / v).

[0012] Furthermore, the TE25S buffer solution comprises: 25mM pH8.0 Tris-HCl, 25mM pH8.0 EDTA, and 10wt% sucrose.

[0013] Furthermore, in step S3, E. coli cell lysis and protein digestion are carried out by adding proteinase K reaction solution to the gel block and reacting at 50°C for 2 h. The proteinase K reaction solution consisted of: 500 mM EDTA, 1% sodium lauroyl sarcosinate, 200 μg / mL proteinase K, and pH adjusted to 8.0.

[0014] Furthermore, in step S3, the gel block after lysis and protein digestion is immersed in a protective solution for long-term preservation.

[0015] Furthermore, the protective solution is a TE10 buffer solution, the components of which are: 10mM pH 8.0 Tris-HCl and 10mM pH 8.0 EDTA.

[0016] Furthermore, in step S4, for different molecular weight ranges, gel blocks containing linear DNA bands of different lengths are separated by specific pulsed-field gel electrophoresis conditions to obtain pulsed-field gel electrophoresis markers. The prepared markers are then tested to determine the integrity and distribution of the marker bands.

[0017] Further, in step S4, pulsed-field gel electrophoresis separation is performed in (0.5-1)×TBE buffer at 7-14°C.

[0018] Furthermore, the pulsed field gel electrophoresis conditions are as follows: depending on the size of the marker bands, the electrophoresis temperature is controlled at 7-14℃, the electric field strength is set to 2-6 V / cm, the pulsed electric field conversion time is 1 second to 500 seconds, the pulsed electric field conversion angle is 106°-120°, and the total electrophoresis time is adjusted between 24 hours and 48 hours.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The pulsed field gel electrophoresis markers prepared by this invention have a wide molecular weight coverage. By combining various Escherichia coli strains containing linear DNA, a series of pulsed field gel electrophoresis markers can be prepared to cover the molecular weight range of 10 kb to 3 Mb, meeting the needs of most pulsed field gel electrophoresis applications.

[0020] 2. The pulsed field gel electrophoresis marker bands prepared by this invention are uniformly distributed. This invention uses the Escherichia coli in vivo linear DNA splicing method, which can splice linear DNA fragments of any size in E. coli. By uniformly selecting E. coli combinations with increasing linear DNA fragment sizes, uniformly distributed DNA bands can be provided within the separation range, which is more uniform than the currently used commercial yeast chromosome markers.

[0021] 3. The pulsed-field gel electrophoresis markers prepared by this invention can be customized according to actual needs. Since linear DNA fragments of any size can be spliced, this invention can adjust the strain combination as needed to prepare pulsed-field gel electrophoresis markers with specific molecular weight ranges or band patterns. For example, after restriction endonuclease XbaI digestion, the genome fragment size of Salmonella typhimurium is mainly distributed between 15Kb and 804Kb. This invention mixes strains 1-19 in Table 1 to prepare pulsed-field gel electrophoresis markers suitable for this range for genome identification after restriction enzyme digestion of Salmonella typhimurium.

[0022] 4. Since E. coli is used as the material to make the marker, the encapsulation and digestion operations are simple and quick, which greatly shortens the production time and reduces the technical difficulty of production. The entire production process only takes 5 hours.

[0023] 5. Using the most commonly used model microorganism Escherichia coli as raw material, the preparation cost, including culture cost and gel block digestion cost, is far lower than other commercial pulsed field gel electrophoresis markers such as yeast chromosome DNA and λ phage DNA tandem ladder markers.

[0024] 6. Because the prepared pulsed-field gel electrophoresis marker DNA fragments are embedded in low-melting-point agarose gel and then immersed in preservation solution, the marker DNA bands have excellent stability and can be stored for a long time. They can be stably stored at 4 ℃ for at least 12 months.

[0025] 7. The present invention forms a gel block by embedding mixed bacterial cells in a low-melting-point agarose gel. The gel block can protect the DNA marker band from shear damage during subsequent processing. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the process for fabricating a pulsed-field gel electrophoresis marker based on Escherichia coli containing large linear DNA fragments in this invention.

[0027] Figure 2The diagram shows the pulsed-field gel electrophoresis standard PFGE Marker-S (14.7-330 Kb) and the pulsed-field gel electrophoresis results in Example 1. In Example 1, A is a schematic diagram of the composition of E. coli strains in PFGE Marker-S (14.7-330 Kb), which consists of nine strains of E. coli containing linear DNA, from smallest to largest: MT-pLW (14.7 Kb), MT-pLC-1 (37 Kb), MT-pLC-2 (64 Kb), MT-PZT (82 Kb), MT-pLC-3 (117 Kb), p6-Liner (153 Kb), p1 (200 Kb), pGXD5 (241 Kb), and pA (330 Kb). In Example 2, B is a pulsed-field gel electrophoresis detection result of PFGE Marker-S.

[0028] Figure 3 The diagram shows the pulsed-field gel electrophoresis (PFGE) standard PFGE Marker-M (200 Kb-1.6 Mb) and the results of PFGE Marker-M. In Example 2, A is a schematic diagram of the composition of E. coli strains in PFGE Marker-M (220 Kb-1.6 Mb), which consists of nine strains of E. coli containing linear DNA, from smallest to largest: p1 (200 Kb), pA (330 Kb), pB (456 Kb), pGXC5 (627 Kb), pGXE3 (813 Kb), pGXD8 (974 Kb), pGXE4 (1.1 Mb), ELC121-pWT1284 (1.2 Mb), and pGXG1 (1.6 Mb). B is a result of PFGE Marker-S pulsed-field gel electrophoresis.

[0029] Figure 4 The diagram shows the pulsed-field gel electrophoresis (PFGE) standard PFGE Marker-L (0.87 Mb-2.8 Mb) and the PFGE results in Example 3. A shows the composition of the E. coli strains in PFGE Marker-L (0.87 Mb-2.8 Mb), consisting of four strains containing linear DNA: ELC121-90-1 (0.87 Mb), ELC121-90-2 (1.2 Mb, 2.8 Mb), pGXG1 (1.6 Mb), and pGXE6 (2.06 Mb). B shows the PFGE Marker-L pulsed-field gel electrophoresis detection results. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0031] In the following embodiments, the linear DNA length and sequence of the *E. coli* strain are shown in Table 1: Table 1. Escherichia coli strains containing linear, ultra-large DNA Among them, p1, pA, and pB are derived from the article Zhong, L., et al., The conjugation-associated linear-BAC iterative assembling (CALBIA) method for cloning 2.1-Mb human chromosomal DNAs in bacteria. . Cell Research, 2025. 35(4): p. 309-312. pGXE4 and pGXG1 are derived from the article Wang T, He F, He T, Lin C, Guan X, Qin Z, Xue X. Reconstruction of a robust bacterial replication module. Nucleic Acids Res. 2024 Oct 14;52(18):11394-11407. MT-pLW: MDS42 E. coli containing a 14.7 kb linear DNA fragment from the BAC plasmid containing the EGFP element and the tos site.

[0032] MT-pLC-1, MT-pLC-2, and MT-pLC-3: each contains clones of deep-sea bacteria with BAC as the backbone. Zhurongbacter thermophilus MDS42 Escherichia coli contains linear DNA fragments from the 56-27923, 56-53836, and 56-106240 regions of the 3DAC genome.

[0033] MT-PZT: Contains clones of deep-sea archaea with BAC as the backbone. Thermococcus eurythermalis MDS42 Escherichia coli contains a linear DNA fragment from the 132822-206866 region of the A501 genome.

[0034] p6-Liner: MDS42 Escherichia coli containing a linear DNA fragment cloned from the 106509911-106656138 region of human chromosome 14 with a BAC backbone.

[0035] pGXD5: Contains clones derived from *Stibulobacterium crescentis* with a BAC backbone. Caulobacter crescentusMDS42 Escherichia coli (NA1000) is a linear DNA fragment from the 982926-1209086 region of the genome.

[0036] pGXC5: Contains clones derived from *Stibulobacterium crescentis* with a BAC backbone. Caulobacter crescentus MDS42 Escherichia coli (NA1000) is a linear DNA fragment from the 428320-1040822 region of the genome.

[0037] pGXE3: Contains clones derived from *Stibulobacterium crescentis* with a BAC backbone. Caulobacter crescentus MDS42 Escherichia coli (NA1000) is a linear DNA fragment from the 3519465-277326 region of the genome.

[0038] pGXD8: Contains clones from *Stibulobacterium crescentis* with a BAC backbone. Caulobacter crescentus MDS42 Escherichia coli (NA1000) is a linear DNA fragment from the 982926-1941341 region of the genome.

[0039] pGXE6: Contains clones derived from *Stipa lunata* with a BAC backbone. Caulobacter crescentus MDS42 Escherichia coli (NA1000) is a linear DNA fragment from the 3936983-1941341 region of the genome.

[0040] ELC121-pWT1284: MDS42 Escherichia coli containing a linear DNA fragment of the genome 1-1209200 region of E. coli cloned with BAC as the backbone.

[0041] ELC121-90-1: MDS42 Escherichia coli containing a linear DNA fragment of the genome 3120122-3978103 of Escherichia coli cloned with OriC+ParABS(RKⅡ) as the backbone (i.e., an active distribution system ParABS with OriC as the origin of replication and RKⅡ plasmid-derived distribution elements).

[0042] ELC121-90-2: An E. coli strain containing a linear DNA fragment from the 1-1209200 region of the genome of E. coli MDS42 cloned with OriC+ParABS(RKⅡ) as the backbone (i.e., an active distribution system ParABS with OriC as the origin of replication and RKⅡ plasmid-derived distribution elements) and a linear DNA fragment from the 1209200-3978103 region of the genome of E. coli MDS42 itself with OriC as the backbone (i.e., OriC as the origin of replication).

[0043] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0044] Example 1 This embodiment provides a method for preparing the pulsed-field gel electrophoresis standard PFGE Marker-S (14.7-330 Kb), the specific steps of which are as follows: S1. Nine Escherichia coli strains containing linear DNA, namely MT-pLW (14.7Kb), MT-pLC-1 (37Kb), MT-pLC-2 (64Kb), MT-PZT (82Kb), MT-pLC-3 (117Kb), p6-Liner (153Kb), p1 (200Kb), pGXD5 (241Kb), and pA (330Kb), were picked from the plate and transferred to 5 mL of LB medium. The medium was incubated overnight at 37°C and 220 rpm to obtain the amplified culture of the nine strains. S2. Take 250 μL of the amplified bacterial culture obtained in step S1 and dilute it to 1 mL. Measure the OD of the diluted solution. λ=600nm The value, multiplied by 4, is the concentration of the amplification culture solution for each strain; S3. Using the concentration of the amplified culture solution of each strain calculated in step S2, take the OD value of each strain. 600 =1 bacterial culture, centrifuged at 7000 rpm for 5 minutes, removed the supernatant to obtain bacterial cells; S4. Resuspend the bacterial cells obtained in step S3 in 1 mL of TE25S solution (25 mM pH 8.0 Tris-HCl, 25 mM pH 8.0 EDTA, 10% sucrose) to obtain a resuspension. S5. Take 200 μL of the resuspension of each of the 9 strains obtained in step S4, vortex and mix them evenly in a 2 ml EP tube, centrifuge at 7000 rpm for 5 minutes, remove the supernatant, and obtain the mixed bacterial cells. S6. Resuspend the mixed bacterial cells obtained in step S5 with 100 μL of TE25s solution, shake to mix evenly, add an equal volume of 2% (w / v) low melting point agarose solution (the low melting point agarose solution is obtained by dissolving low melting point agarose powder purchased from Sangon Biotech A600015 in TE25S buffer, which has been preheated and kept equilibrated in a 50°C water bath), mix by blowing and aspiration, pour into a pulsed field gel electrophoresis mold, and cool at 4°C for 30 minutes to obtain a gel block; S7. Add 10 mL of proteinase K reaction solution (pH 8.0, 500 mM EDTA, 1% sodium lauroyl sarcosinate, 200 μg / mL proteinase K) to each 1 mL of gel block obtained in step S6 and digest at 50 °C for 2 hours. Take 2 mL of TE10 solution (10 mM pH 8.0 Tris-HCl, 10 mM pH 8.0 EDTA) and wash the digested gel block 3 times. Gently shake at room temperature for 30 minutes each time. After washing, store the gel block in a protective solution (TE10 buffer, the components of which are: 10 mM pH 8.0 Tris-HCl, 10 mM pH 8.0 EDTA) for a long time. S8. Separation was performed using a CHEF Mapper A7 pulsed-field gel electrophoresis system from Zhongke Yongyi at 14°C in 0.5*TBE buffer, with the pulsed electric field switching angle set to 120°. o The pulse voltage was set to 6V / cm, the pulse electric field conversion time was 1s-25s, and the electrophoresis time was 24 hours. Pulsed field gel electrophoresis standard PFGE Marker-S with a separation range of 14.7-330 Kb was obtained.

[0045] The electrophoresis results of the pulsed-field gel electrophoresis standard PFGE Marker-S are as follows: Figure 2 As shown in B, Figure 2 The leftmost element in B is a commercially available λ phage DNA tandem ladder marker, with each band having a defined size. The accuracy of the band size of the pulsed-field gel electrophoresis standard PFGE Marker-S made from E. coli containing linear DNA according to this invention can be determined by comparing it with that of the standard. The PFGE Marker-S pulsed-field gel electrophoresis standard made from E. coli containing linear DNA according to this invention has clear, bright, and uniformly distributed bands, with a separation range of 14.7-330 Kb, making it suitable for molecular weight calibration and quality control in pulsed-field gel electrophoresis experiments within this molecular weight range.

[0046] Example 2 This embodiment provides a method for preparing the pulsed-field gel electrophoresis standard PFGE Marker-M (200 Kb-1.6 Mb). Except for step S1, which involves culturing nine E. coli strains containing linear DNA, namely p1 (200 Kb), pA (330 Kb), pB (456 Kb), pGXC5 (627 Kb), pGXE3 (813 Kb), pGXD8 (974 Kb), pGXE4 (1.1 Mb), ELC121-pWT1284 (1.2 Mb), and pGXG1 (1.6 Mb), the pulsed-field gel electrophoresis conditions in step S8 are as follows: the pulsed electric field switching angle is set to 120°. oThe voltage was set to 6V / cm, the pulse electric field conversion time was 60s-120s, and the electrophoresis time was 24 hours; the remaining steps were the same as in Example 1.

[0047] The results of pulsed-field gel electrophoresis are attached. Figure 3 As shown in B, Figure 3 The leftmost element in B is a commercially available Saccharomyces cerevisiae chromosome DNA marker, with each band having a defined size. The accuracy of the band size of the pulsed-field gel electrophoresis standard PFGE Marker-M, made from E. coli containing linear DNA according to this invention, can be determined by comparing it with that standard. The PFGE Marker-M pulsed-field gel electrophoresis standard made from E. coli containing linear DNA according to this invention has clear, bright, and uniformly distributed bands, with a separation range of 200 Kb-1.6 Mb, making it suitable for molecular weight calibration and quality control in pulsed-field gel electrophoresis experiments within this molecular weight range.

[0048] Example 3 This embodiment provides a method for preparing the pulsed-field gel electrophoresis standard PFGE Marker-L (0.87Mb-2.8Mb). In step S1, four E. coli strains containing linear DNA—ELC121-90-1 (0.87Mb), ELC121-90-2 (1.2Mb, 2.8Mb), pGXG1 (1.6Mb), and pGXE6 (2.06Mb)—are cultured. In step S4, a CHEF Mapper A7 pulsed-field gel electrophoresis instrument is used for separation in 1*TAE buffer at 7°C. The pulsed-field gel electrophoresis conditions are set with the pulse electric field switching angle at 106°C. o The voltage was set to 3V / cm, the pulse electric field conversion time was 500s, and the electrophoresis time was 48 hours; the remaining steps were the same as in Example 1.

[0049] The results of pulsed-field gel electrophoresis are attached. Figure 4 As shown in B, Figure 4 The leftmost element in B is a commercially available fission yeast chromosome DNA marker, with each band having a defined size. The accuracy of the band size of the pulsed-field gel electrophoresis standard PFGE Marker-L, made from E. coli containing linear DNA according to this invention, can be determined by comparison with it. The PFGE Marker-L pulsed-field gel electrophoresis standard made from E. coli containing linear DNA according to this invention has clear, bright, and uniformly distributed bands, with a separation range of 0.87Mb-2.8Mb, making it suitable for molecular weight calibration and quality control in pulsed-field gel electrophoresis experiments within this molecular weight range.

[0050] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments, characterized in that... Includes the following steps: S1. Culture and mix various E. coli strains containing linear DNA to obtain mixed bacterial cells. Among them, the linear DNA contained in each type of Escherichia coli is of different length; S2. The mixed bacterial cells obtained in step S1 are embedded in a low-melting-point agarose gel to obtain a gel block; S3. Perform E. coli cell lysis and protein digestion on the gel block obtained in step S2 to obtain a gel block containing linear DNA bands of different lengths. S4. The gel block containing linear DNA bands of different lengths obtained in step S3 is separated by pulsed-field gel electrophoresis, which is the pulsed-field gel electrophoresis marker.

2. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 1, characterized in that, In step S1, the length of the linear DNA in the E. coli containing linear DNA ranges from 10 Kb to 3000 Kb.

3. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 2, characterized in that... In step S1, the mixed bacterial cells are selected from any two or more combinations of MT-pLW, MT-pLC-1, MT-pLC-2, MT-PZT, MT-pLC-3, p6-Liner, p1, pGXD5, pA, pB, pGXC5, pGXE3, pGXD8, pGXE4, ELC121-pWT1284, pGXG1, ELC121-90-1, ELC121-90-2, and pGXE6.

4. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 1, characterized in that, In step S1, the E. coli containing linear DNA is obtained by the following method: A series of E. coli strains containing linear DNA of different lengths were obtained by custom splicing through conjugation transfer and homologous recombination. High-density cells of each strain were obtained by amplification culture.

5. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 4, characterized in that... In step S1, various types of E. coli containing linear DNA are subjected to overnight amplification culture to obtain bacterial cells, and the OD of each bacterial cell is measured. 600 OD of each strain 600 Mix the bacterial cells evenly in quantities of 0.1-1 to obtain mixed bacterial cells.

6. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 1, characterized in that, In step S2, the low-melting-point agarose gel is a mixture of low-melting-point agarose powder and TE25S buffer, and the final concentration of the low-melting-point agarose powder is 1-2% (w / v).

7. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 6, characterized in that, The TE25S buffer solution consists of: 25mM pH8.0 Tris-HCl, 25mM pH8.0 EDTA, and 10wt% sucrose.

8. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 1, characterized in that, In step S3, E. coli cell lysis and protein digestion are carried out by adding proteinase K reaction solution to the gel block and reacting at 50°C for 2 h. The proteinase K reaction solution consisted of: 500 mM EDTA, 1% sodium lauroyl sarcosinate, 200 μg / mL proteinase K, and pH adjusted to 8.

0.

9. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 1, characterized in that, In step S3, the gel blocks after lysis and protein digestion are immersed in a protective solution for long-term preservation. The protective solution is TE10 buffer, and the components of the TE10 buffer are: 10mM pH8.0 Tris-HCl and 10mM pH8.0 EDTA.

10. The method for preparing pulsed-field gel electrophoresis markers based on *E. coli* containing large linear DNA fragments according to claim 1, characterized in that, In step S4, pulsed-field gel electrophoresis separation is performed in (0.5-1)×TBE buffer at 7-14℃. The pulsed-field gel electrophoresis conditions are as follows: depending on the size of the marker band, the electrophoresis temperature is controlled at 7-14℃, the electric field strength is set to 2-6 V / cm, the pulse electric field switching time is 1 second to 500 seconds, the pulse electric field switching angle is 106°-120°, and the total electrophoresis time is adjusted between 24 hours and 48 hours.

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