Escherichia coli W2 for separating intestinal escherichia coli

Abstract

The invention relates to an intestinal Escherichia coli isolated bacterium E.coli W2 which is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC 66115, and the preservation date is April 8, 2025. The isolated strain is Escherichia coli, the sequence of the Escherichia coli is different from that of common Escherichia coli, and the Escherichia coli is a nematode intestinal source natural isolated strain. Compared with Escherichia coli E.coli Nissle 1917 commonly used in related animal intestinal tract research, the strain has higher plasmid transformation capacity, the transformation efficiency is improved by 2.2 times, a higher colonization level and longer colonization time can be kept in the nematode intestinal tract colonization process, the colonization level of W2 after the nematode intestinal tract colonization is 144 hours is as high as 1769.9 times compared with E.coli Nissle 1917, and the nematode intestinal tract colonization level is as high as 1769.9 times compared with E.coli Nissle 1917. And the strain can become a candidate strain for related research of nematode intestinal tracts.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology engineering, and particularly relates to an Escherichia coli strain with high plasmid transformation ability and high colonization level, which can colonize in the intestine of Caenorhabditis elegans for a long time and is used for research related to the nematode intestine. Background Art

[0002] In the field of biology, nematodes are model organisms widely used in the study of many biological processes. Nematodes feed on bacteria, and the ingested bacteria are digested in the intestine to provide nutrients for nematodes, which makes nematodes represent a very easy-to-genetically-manipulate model biological system for studying the direct / indirect effects of diet, including host-microbe interactions. Among them, Caenorhabditis elegans is a powerful tool for studying host-microbe interactions. Escherichia coli, due to its biological characteristics and the convenience of genetic manipulation, helps to explore complex problems in nematode and host-microbe biology.

[0003] Currently, the engineering Escherichia coli commonly used in intestinal research are mainly E. coli Nissle 1917 and E. coli MG1655, but their colonization levels and colonization times in the nematode intestine are relatively low. Summary of the Invention

[0004] The present invention aims to solve these problems in the prior art by providing a new nematode intestinal origin Escherichia coli strain, which shows better performance in plasmid transformation efficiency and colonization level. This new strain will provide a more effective and reliable tool for research related to the intestine of Caenorhabditis elegans.

[0005] To solve the above technical problems, the present invention provides an intestinal origin Escherichia coli W2 isolated from the intestine of Caenorhabditis elegans at the L4 life cycle stage. This strain has good plasmid transformation ability and high colonization level, and can contribute to research related to the nematode intestine.

[0006] To achieve the above invention object, the technical solution implemented by the present invention is as follows:

[0007] Isolate intestinal origin Escherichia coli from the intestinal lysate of Caenorhabditis elegans, screen through LB and Escherichia coli chromogenic plates, and perform PCR amplification on the 16S rRNA gene sequence;

[0008] Compare the 16S rRNA gene sequence obtained by the above PCR amplification with the sequences of common Escherichia coli and other related species to construct a MEGA phylogenetic tree;

[0009] To compare whether there are advantages in the gene manipulation functions of Escherichia coli isolated from the intestine, the transformation efficiency of the strains was first detected. The plasmid J23101-sfgfp@pSB3K3 with the expression of the green fluorescent protein gene existing in the laboratory was transferred into the Escherichia coli to be tested through bacterial transformation to form a transformed engineering bacterium. Different dilutions were made and the bacteria were spotted on LB and LB + kanamycin solid plates, and the transformation efficiency was calculated based on the number of single colonies.

[0010] To compare the colonization level and duration of Escherichia coli isolated from the intestine in Caenorhabditis elegans, the λ-Red homologous recombination gene editing technology was used to insert the green fluorescent protein gene and the kanamycin resistance gene into the Escherichia coli genome, enabling the strain to be stably expressed in the nematode and facilitating subsequent observation and screening. After feeding the three knockout strains of E. coli W2ΔlacZ::sfgfp KmR, E. coli Nissle 1917ΔlacZ::sfgfp KmR, and E. coli MG1655ΔlacZ::sfgfp KmR to the nematodes until they reached the L4 stage, the nematodes were transferred to a heat-inactivated E. coli OP50 NGM solid medium for cultivation. At four time points of 24h, 48h, 96h, and 144h after feeding respectively, the nematodes were collected. The intestinal lysates of the nematodes were diluted at different concentration gradients and spotted on LB + kanamycin solid plates, and the CFU carried by each nematode was estimated based on the number of single colonies to compare the colonization levels of different Escherichia coli.

[0011] Beneficial effects: To find Escherichia coli that can colonize the nematode intestine for a long time, the present invention isolated Escherichia coli from the nematode intestine by simulating the rich microbial flora of the nematode in the wild. Secondly, in order to enhance the gene transformation efficiency of Escherichia coli, different methods for preparing competent cells and transformation conditions were used to affect the transformation effect. In order to facilitate the tracking research and screening of Escherichia coli in the nematode intestine in vivo, Escherichia coli gene knockout bacteria were constructed by the λ-Red homologous recombination gene editing technology so that their genomes carry the green fluorescent protein gene and the kanamycin resistance gene.

[0012] The above method steps of the present invention comprehensively utilize the advantages of the green fluorescent protein gene and nematode intestinal Escherichia coli. Through MEGA phylogenetic tree, transformation efficiency, and colonization level experiments, a strain of nematode intestinal Escherichia coli E. coli W2 with high efficiency in all aspects was finally screened. Compared with the model Escherichia coli commonly used in nematode intestinal research in existing articles, this bacterium has a higher transformation efficiency and a higher colonization level in the nematode intestine, and thus has more advantages. Description of the Drawings

[0013] The present invention will be further described below in conjunction with the drawings and embodiments.

[0014] Figure 1 This is the MEGA phylogenetic tree diagram of the nematode intestine-derived isolate of the present invention and the 16S rRNA gene sequence of the studied Escherichia coli.

[0015] Figure 2 This is the comparison diagram of the plasmid transformation effects of the nematode intestine-derived isolate of the present invention and E. coli Nissle 1917 and E. coli MG1655.

[0016] Figure 3 This is the comparison diagram of the colony-forming unit (CFUs) data formed by the nematode intestine-derived isolate of the present invention and E. coli Nissle 1917 and E. coli MG1655 in the nematode intestine. Detailed implementation mode

[0017] Main materials:

[0018] Wild-type Caenorhabditis elegans Bristol strain, N2

[0019] 2×Phanta Max Master Mix was purchased from Nanjing Novoprotein Science and Technology Co., Ltd.

[0020] 5-Fluorouracil (CAS: 51-21-8) was purchased from Sigma-Aldrich, USA

[0021] E. coli chromogenic medium was purchased from Qingdao Haibo Biotech Co., Ltd.

[0022] Sterile PBS buffer was purchased from Sevier Biotech Co., Ltd.

[0023] Triton X-100 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0024] Greiner 96μClear flat bottom microplate was purchased from Guangzhou Dingguo Biotechnology Co., Ltd.

[0025] Other materials not specified were obtained through regular commercial purchases.

[0026] 50 mg / mL kanamycin stock solution: 0.5 g kanamycin, add 10 mL ddH2O, filter with a 0.22 μM filter membrane, aliquot into 1.5 centrifuge tubes, and store at -20 °C.

[0027] 5 mg / mL cholesterol: 25 mg cholesterol, add 50 mL absolute ethanol, filter with a 0.22 μM filter membrane, and store at 4 °C.

[0028] LB solid medium: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 20 g agar powder, add ultrapure water to 1 L;

[0029] LB + Kanamycin solid medium: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 20 g agar powder, add ultrapure water to 1 L, sterilize and cool down to 45 °C, add 50 mg / mL kanamycin solution at a ratio of 1:1000.

[0030] TB liquid medium: 12 g tryptone, 24 g yeast extract, 17 mM KH2PO4, 72 mM K2HPO4, 0.4% glycerol, add ultrapure water to 1 L;

[0031] NGM solid medium: 3 g sodium chloride, 2.5 g tryptone, 17 g agar powder, 25 mL (1 M) potassium phosphate solution, 1 mL (1 M) calcium chloride dihydrate solution, 1 mL (1 M) magnesium sulfate heptahydrate solution, and 1 mL (5 mg / mL) cholesterol, add ultrapure water to 1 L.

[0032] NGM + 5 - Fluorouracil solid medium: 3 g sodium chloride, 2.5 g tryptone, 17 g agar powder, 25 mL (1 M) potassium phosphate solution, 1 mL (1 M) calcium chloride dihydrate solution, 1 mL (1 M) magnesium sulfate heptahydrate solution, and 1 mL (5 mg / mL) cholesterol, add ultrapure water to 1 L, sterilize and cool down to 45 °C, add 20 mM 5 - fluorouracil solution at a ratio of 1:1000.

[0033] M9 buffer: 5 g sodium chloride, 3 g KH2PO4, 6 g Na2HPO4, add ultrapure water to 1 L, sterilize and cool down to 45 °C, add 1 mL (1 M) magnesium sulfate heptahydrate solution.

[0034] Example 1: Screening of Escherichia coli from Caenorhabditis elegans intestine and construction of MEGA phylogenetic tree

[0035] (1) Cultivation of Caenorhabditis elegans

[0036] First, transfer L4-stage nematodes to NGM plates inoculated with E. coli OP50 and incubate them for 24 h. E. coli OP50 serves as their food source [Girard L R, Fiedler T J, Harris T W, et al. WormBook: the online review of Caenorhabditis elegans biology [J]. Nucleic acids research, 2007, 35 (Database issue): D472-475.]. Let them reach the stage where the adult worms' bellies are full of a large number of eggs. Add M9 buffer to the NGM plates containing a large number of adult nematodes full of eggs for collection. Dissolve late-pregnant adult nematodes with bleach (1 mL of 4 M sodium hydroxide, 1.25 mL of sodium hypochlorite, 7.75 mL of ddH2O). This process will release the eggs, and then the eggs are collected to obtain synchronized L1-stage larvae. Feed the L1 nematodes with a simulated wild rich microbial flora using rotten apples, rotten bananas, and yogurt [The method for constructing the microbial flora refers to He YJ, Hao F R, Fu H R, et al. N-glycosylated intestinal protein BCF-1 shapes microbial colonization by binding bacteria via its fimbrial protein [J]. Cell Reports, 2023, 42(1)] until the L4 stage, and then collect the nematodes.

[0037] (2) Isolation and identification of intestinal-source Escherichia coli

[0038] After washing the L4 nematodes 3-5 times with M9 buffer, wash them 2 times with M9 buffer containing 25 mM levamisole (levamisole inhibits pharyngeal pumping and defecation) (LM buffer), then wash them 2 times with LM buffer containing 100 μg / mL gentamicin (to clean the bacteria outside the nematodes), and then wash them three times separately with M9 buffer. Put the washed nematodes into a 1.5 mL centrifuge tube containing 100 μL of PBS with 1% Triton X-100, add sterilized steel beads, and grind them on a grinder (60 Hz, 1 min, repeat 9 times) to make the mother liquor of the nematode intestinal lysate. Pipette 10 μL of the mother liquor and add sterile PBS buffer to 1 mL to obtain a 10 -1 dilution, and so on to obtain 10 -2 -10 -4Diluent. Take 100 μL and coat it on the LB solid medium, and incubate it overnight at 37°C in an incubator. Pick a single colony with a round and neat edge, smooth surface, semi-transparent, and small protrusions similar to Escherichia coli. After streaking and separating on the LB solid medium twice, pick a single colony and streak it on the Escherichia coli chromogenic solid medium to judge the bacterial genus. If the single colony is blue, it is identified as Escherichia coli.

[0039] (3) Construction of the MEGA phylogenetic tree

[0040] Pick a single colony of Escherichia coli, use the primers in Table 1 for amplification in the PCR reaction system in Table 2, and a 16S rRNA gene fragment of about 1500 bp is amplified by PCR.

[0041] Table 1 PCR amplification primers for the identification of nematode intestinal Escherichia coli

[0042]

[0043] Table 2 PCR reaction system (50 μL)

[0044]

[0045] Table 3 PCR amplification process

[0046]

[0047] After the reaction is terminated, it is purified and recovered by 1% agarose gel electrophoresis for standby and subjected to Sanger sequencing (Sangon Biotech Co., Ltd., Shanghai).

[0048] Among them, the neighbor-joining method (NJ) of the MEGA software is used to compare the sequences of the isolated nematode intestinal Escherichia coli with the Escherichia coli commonly used in the study of the intestine, and it is found that there are differences in the sequences, such as Figure 1 .

[0049] After the above cultivation and identification, one of the isolated strains was classified and named as nematode intestinal Escherichia coli E. coli W2.

[0050] The nematode intestinal Escherichia coli E. coli W2 (Escherichia coli W2) was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 8, 2025. The deposit location is Guangzhou, Guangdong Province, and the deposit number is GDMCC 66115.

[0051] Example 2: Plasmid transformation efficiency of nematode intestinal Escherichia coli and common Escherichia coli in self-made competent cells

[0052] The plasmid J23101-sfgfp@pSB3K3 in the laboratory has been constructed based on the common parts library of iGEM (the main components of this plasmid, such as J23101, pSB3K3, and sfgfp, can be found at https: / / parts.igem.org, and then assembled by oneself). This plasmid was transformed into the self-prepared competent cells of each Escherichia coli. It should be noted that other plasmids can also be used to evaluate the plasmid transformation efficiency of Escherichia coli, as long as the plasmids used can continuously express fluorescent proteins and resistance genes. The use of plasmid J23101-sfgfp@pSB3K3 is only because it is convenient to obtain. Therefore, the specific construction method of this plasmid is not introduced in detail. Those skilled in the art can, according to the description of the present invention, independently use other plasmids that can continuously express fluorescent proteins and resistance genes to conduct experiments on evaluating the transformation efficiency of Escherichia coli.

[0053] Among them, the competent cells were prepared by the CaCl2 method. A small amount was picked from the preserved strain and streaked on an LB plate, and cultured at 37°C. A single colony on the plate was picked into 5 mL of LB and cultured with shaking at 37°C and 180 rpm for 17 h. The overnight culture was diluted 400-fold into 200 mL of fresh LB and cultured with shaking at 37°C and 180 rpm until OD 600 = 0.3 - 0.4. The culture was transferred to a 50 mL centrifuge tube and immediately placed on ice for 20 min. The centrifuge tube was placed in a centrifuge pre-cooled at 4°C, and centrifuged at 3000g for 8 min. The supernatant was discarded, 10 mL of pre-cooled 50 mM CaCl2 was added, and the cells were gently resuspended on ice. The previous centrifugation step was repeated, the cells were collected, the supernatant was discarded again, 10 mL of pre-cooled 50 mM CaCl2 was added, and the cells were gently resuspended on ice and placed on ice for 30 min. Then, it was centrifuged at 3000g for 6 min again, and the supernatant was discarded. 2 mL of 50 mM CaCl2 and 15% glycerol were added to each 50 mL centrifuge tube, and the cells were gently resuspended, placed on ice for 2 h, then aliquoted into 1.5 mL centrifuge tubes, quickly frozen in liquid nitrogen, and stored at -80°C.

[0054] Among them, the transformation of the plasmid was carried out by heat shock transformation. The plasmid J23101-sfgfp@pSB3K3 was transferred into 100 μL of Escherichia coli competent cells with a final concentration of 1 μg / mL. According to the conventional transformation procedure, the plasmid was added to the competent cells and placed on ice for 30 min, heat shocked at 42°C for 90 s, immediately placed on ice for 3 min, and then 900 μL of fresh LB was added and cultured at 37°C for 1 h for recovery. The transformation products were serially diluted, and 3 μL of each dilution was spotted on LB and LB + kanamycin solid plates, with three replicates, and cultured inverted at 37°C for 17 h. The transformation efficiency % was calculated according to the number of colonies at the same concentration: Transformation efficiency % = (Number of single colonies on the LB + kanamycin solid plate) / (Number of single colonies on the LB solid plate × dilution factor) × 100%.

[0055] The transformation efficiency of Escherichia coli was calculated and the results are shown in Table 4. The transformation efficiency of E. coli W2 isolated from nematode intestine was 2.2 times higher than that of the commonly used model E. coli Nissle 1917.

[0056] Table 4 Transformation efficiency of E. coli isolated from nematode intestine and commonly used model E. coli

[0057]

[0058] Experimental Example 3: Comparison of the colonization levels of E. coli isolated from nematode intestine and commonly used model E. coli in the nematode intestine

[0059] Using the λ-Red recombination technique, gene knockout bacteria were constructed from the isolated E. coli W2, E. coli Nissle 1917, and E. coli MG1655, and were named E. coli W2ΔlacZ::sfgfp KmR, E. coli Nissle 1917ΔlacZ::sfgfp KmR, and E. coli MG1655ΔlacZ::sfgfp KmR, respectively.

[0060] First, a single colony was picked from a freshly streaked E. coli OP50 plate and inoculated into 400 mL of LB medium. The culture was shaken at 250 rpm at 37 °C for 16 - 22 hours. The cultured E. coli OP50 (200 mL) was concentrated to 100 mL (1 / 2 volume) by centrifugation (2000 g, 15 min). The pellet was vortexed to resuspend the cells. The concentrated E. coli OP50 was heat-inactivated in a 90 °C water bath for 120 minutes. 500 μL of heat-inactivated OP50 (HK-OP50) was spread on an LB plate to observe colony growth. The plate was incubated at 37 °C for 16 - 22 hours. No bacterial growth confirmed successful heat inactivation. 0.3 mg / mL riboflavin (vitamin B2) was spread on NGM plates to maintain normal nematode development. Subsequently, 300 μL of HK-OP50 was spotted onto NGM-5-fluorouracil (5-Fu) plates to prepare HK-OP50-NGM-5fu medium.

[0061] Pick one single colony from the streaked plates of E. coli W2ΔlacZ, E. coli Nissle 1917ΔlacZ, and E. coli MG1655ΔlacZ, and inoculate them into 5 mL of LB liquid medium. Incubate the cultures at 37 °C with shaking at 250 rpm for 16 - 22 hours. Dilute 0.5 mL of the three gene knockout bacteria cultures into 50 mL of fresh LB. Incubate the diluted bacteria at 37 °C until the OD600 reaches about 1. Collect the bacterial cultures, concentrate the three cultures to 25 mL (1 / 2 volume), and vortex to resuspend the bacteria. Spot 300 μL of the bacterial cultures onto 6 cm NGM plates to prepare NGM media with E. coli W2ΔlacZ, E. coli Nissle 1917ΔlacZ, and E. coli MG1655ΔlacZ for feeding nematodes.

[0062] Centrifuge synchronized L1-stage nematodes in a 15 mL tube to remove the M9 medium. Resuspend the synchronized L1-stage nematodes in 500 μL of M9, then take 10 μL of the L1-stage nematodes and place them on a microscope slide for counting. The ideal number of nematodes is 100 - 200 per 10 μL. Inoculate 40 μL of the synchronized L1-stage nematodes onto NGM media with E. coli W2ΔlacZ::sfgfpKmR, E. coli Nissle 1917ΔlacZ::sfgfp KmR, and E. coli MG1655ΔlacZ::sfgfp KmR, and incubate at 20 °C for 48 h until the L4 stage. Wash the L4-stage nematodes 5 - 6 times with M9 medium, transfer them to HK-OP50-NGM-5fu medium, and transfer them to fresh HK-OP50-NGM-5fu medium daily. Collect the nematodes at the time points of 24 h, 48 h, 96 h, and 144 h of feeding, and lyse the nematode intestines.

[0063] Wash the nematodes 3 times with M9 buffer at the selected time points, and then wash the washed nematodes 3 times with phosphate-buffered saline (PBS) to remove the excess M9 buffer. Transfer them to a 1.5 mL centrifuge tube containing 100 μL of PBS with 1% Triton X-100 and 18% iodixanol (CBI).

[10] CBI can keep the nematodes evenly suspended for easy nematode counting. Take 10 μL for counting to estimate the number of the remaining 90 μL of nematodes. Grind the remaining 90 μL using a grinder to break the nematode cells and release the bacteria in the intestines. Determine the colony-forming units (CFUs) of the three strains in the Caenorhabditis elegans intestines, and calculate the CFUs per nematode intestine according to the formula.

[0064]

[0065] Each condition was repeated biologically at least 6 times, and the results showed that the colonization level of E. coli W2 in the intestine of Caenorhabditis elegans was much higher than that of E. coli Nissle 1917 and E. coli MG1655, as Figure 3 .

[0066] Figure 1 : MEGA phylogenetic tree diagram of the 16S gene sequences of the nematode intestinal isolate E. coli W2 of the present invention, the common E. coli E. coli MG1655, E. coli Nissle1917, and other related species. The 16S gene sequences of other species outside E. coli W2 are from the NCBI database, and Enterococcus faecium strain LMG 11423 is used as an outgroup.

[0067] Based on the 16S rRNA sequences of 21 bacteria, the phylogenetic tree constructed by the neighbor-joining method (NJ) showed that the similarity between the strains E. coli W2 and E. coli R1Q1 was 84%.

[0068] Figure 2 : Comparison diagram of the plasmid transformation effects of the nematode intestinal isolate E. coli W2 of the present invention, the common model E. coli E. coli MG1655, and E. coli Nissle1917. (a) Diagram of the spotted plate area of the bacterial solution after different concentrations of dilution after bacterial transformation, where 0 represents the mother solution, -1 to -12, etc. represent dilution multiples of 10 -1 ~10 -12 etc.; (b) Number of colony distributions of E. coli MG1655, E. coli Nissle 1917, and E. coli W2 on LB + kanamycin solid medium; (c) Number of colony distributions of E. coli MG1655, E. coli Nissle 1917, and E. coli W2 on LB solid medium; (d) Table of dilution multiples, single colony statistics, and transformation efficiency of E. coli MG1655, E. coli Nissle 1917, and E. coli W2 on LB + kanamycin solid medium and LB solid medium.

[0069] Calculated based on the number of colonies at the same concentration. Transformation efficiency % = (number of single colonies on LB + kanamycin solid plate) / (number of single colonies on LB solid plate × dilution multiple) × 100%. By calculating the transformation efficiency of E. coli, it was found that the transformation efficiency of the nematode intestinal E. coli E. coli W2 was 2.2 times higher than that of the common model E. coli E. coli Nissle 1917.

[0070] Figure 3 : Comparative graph of the colonization levels of the E. coli W2, a bacterium isolated from the intestine of nematodes of the present invention, with the commonly used E. coli, E. coli Nissle 1917, and E. coli MG1655 in the intestine of Caenorhabditis elegans. (a) Schematic diagram of spotting the bacterial liquid after dilution to different concentrations after lysing the nematode intestine, where 0 represents the stock solution, -1 to -4, etc. represent dilution multiples such as 10 -1 ~10 -4 and so on; (b) Colony distribution numbers of E. coli W2, E. coli Nissle 1917, and E. coli MG1655 on LB + kanamycin solid medium; (c) CFU statistics of E. coli W2, E. coli Nissle 1917, and E. coli MG1655 on LB + kanamycin solid medium; (d) Comparative graph of CFU data of E. coli W2, E. coli Nissle 1917, and E. coli MG1655 in the intestine of Caenorhabditis elegans.

[0071] The collected intestinal lysate was diluted to an appropriate dilution gradient for spotting bacteria, and the colony numbers of each type of E. coli were counted, so as to calculate CFU. After data comparison, the results were obtained. The colonization level of E. coli W2 was 1769.9 times that of E. coli Nissle 1917 144 hours after colonization in the nematode intestine.

[0072] As mentioned above, it is only a preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. An intestinal Escherichia coli E. coli W2 isolated from the intestine of Caenorhabditis elegans, characterized in that, The preservation number of this bacterium in the Guangdong Provincial Microbial Culture Collection Center is GDMCC 66115, and the preservation date is April 8, 2025.

2. The Caenorhabditis elegans intestinal source Escherichia coli according to claim 1, characterized in that, The said strain has a high plasmid transformation ability and a high colonization efficiency; compared with Escherichia coli Nissle 1917, the plasmid transformation efficiency is increased by 2.2 times; after 144 h of colonization in the nematode intestine, the colonization level of W1 is 1769.9 times that of Escherichia coli Nissle 1917.

3. The intestinal origin Escherichia coli E. coli W2 according to claim 1, characterized in that, The cultivation method of this bacterium includes the following steps: pick a small amount from the preserved Escherichia coli W2 and streak it on an LB solid plate, and the cultivation temperature is 37 °C. Among them, on the LB solid plate, white, semi-transparent, round and smooth colonies can be formed after cultivation at 37 °C.

4. Use of the intestinal Escherichia coli E. coli W2 according to any one of claims 1 to 3 in animal intestinal research.

5. The application according to claim 4, wherein The said animal intestinal research includes plasmid conversion research.

6. The application according to claim 4, characterized in that The said animal intestinal research includes microbial colonization research.

7. The application according to claim 4, wherein The said animal is Caenorhabditis elegans.

8. The application according to claim 4, wherein The said use is to study the host-microbe interaction mechanism under different environmental conditions.

9. The application according to claim 8, wherein The said host-microbe interaction mechanism includes analyzing multiple metabolic pathways to reveal the biological regulatory functions in the host-microbe metabolic process.

10. The application according to claim 8, characterized in that, The said host-microbe interaction mechanism includes using the advantage that this bacterium can colonize for a long time as the chassis cell of a microbial sensor to monitor biomarkers in vivo.

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