Murine Escherichia coli with strong gene manipulability and application of mouse Escherichia coli

Abstract

The invention discloses murine Escherichia coli with strong gene manipulability and application of the murine Escherichia coli, the murine Escherichia coli separates a strain G21229, the preservation number is GDMCC.No: 65288, and the preservation date is October 18, 2024. The isolated strain disclosed by the invention is Escherichia coli, the sequence of the Escherichia coli is different from that of common Escherichia coli, and the Escherichia coli is a mouse-derived natural isolated strain. Compared with escherichia coli E.coli Nissle 1917 commonly used in mouse intestinal research, the strain has higher plasmid transformation capacity, the transformation efficiency is improved by 13 times, higher plasmid stability can be kept in the passage process, and the plasmid fluorescence failure rate is about 1 / 2 of that of E.coli Nissle 1917 and E.coli MG1655, so that the strain has higher gene manipulation capacity, and can be used for preparing a recombinant plasmid for preparing the recombinant plasmid. The strain is hopeful to become a candidate strain for related research of mouse intestinal tracts.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to an Escherichia coli strain with high-efficiency plasmid transformation ability and low plasmid failure rate, which can be used for mouse intestinal related research. Background Art

[0002] In the field of microbiology and molecular biology, Escherichia coli is widely used as a host bacteria for gene cloning and expression due to its easy culture and genetic manipulation. Plasmid transformation technology of Escherichia coli is a basic technology in genetic engineering, which allows the introduction of foreign DNA into bacterial cells to achieve gene cloning and expression.

[0003] In mouse intestinal research, the use of E. coli as a model microorganism can provide important insights into the adaptability of the intestinal environment. In order to enhance the gene transformation efficiency of E. coli, current research mainly uses different competent cell preparation methods. 1,2 and transformation conditions 3 To improve the plasmid stability of E. coli, the main method is to change the growth environment of the strain. 4 to affect the stability of the plasmid.

[0004] E. coli Nissle 1917 is currently commonly used to study the mouse intestine 5 and E. coli MG1655 6 It has limited colonization ability in the mouse intestine and cannot exist stably for a long time.

[0005] References:

[0006] 1Tu,Q.et al.Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.Scientific Reports 6,(2016).

[0007] 2Liu,X.et al.The Study on the factors affecting transformationefficiency of E-coli competent cells. Pakistan Journal of PharmaceuticalSciences 27,679-684(2014).

[0008] 3Chan, W.-T., Verma, CS, Lane, DP&Gan, SK-EA comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Bioscience Reports 33, 931-U172, (2013).

[0009] 4Wein, T., Huelter, NF, Mizrahi, I. & Dagan, T. Emergence of plasmidstability under non-selective conditions maintains antibioticresistance. Nature Communications 10, (2019).

[0010] 5Russell, BJet al. Intestinal transgene delivery with native E. colichassis allows persistent physiological changes. Cell 185, 3263-+, (2022).

[0011] 6Chang, D.-E. et al. Carbon nutrition of Escherichia coliin the mouseintestine. Proceedings of the National Academy of Sciences of the United States of America 101, 7427-7432, (2004). Summary of the invention

[0012] The present invention aims to provide a new mouse-derived Escherichia coli strain, which has better performance in plasmid transformation efficiency and plasmid stability, and can theoretically colonize the mouse intestine. This new strain can provide a more effective and reliable tool for mouse intestinal related research.

[0013] In order to solve the above technical problems, the present invention provides a strain of mouse Escherichia coli isolated from the colorectal contents of 22-week-old C57BL / 6 female mice. The strain has good plasmid transformation ability, low plasmid failure rate during the passage process, and can be stably inherited, making contributions to mouse intestinal related research.

[0014] The mouse-derived Escherichia coli isolate E. coli G21229 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC.No65288 and the deposit date October 18, 2024.

[0015] The present invention separates mouse-derived Escherichia coli from mouse intestinal feces, screens them using LB and Escherichia coli colorimetric plates, and amplifies the 16S sequence using PCR;

[0016] Comparing the 16S sequence obtained by PCR amplification with commonly used Escherichia coli sequences and other closely related bacterial species to construct a MEGA evolutionary tree;

[0017] In order to compare whether the gene manipulation function of mouse-derived E. coli has advantages, the transformation efficiency of the strain was first tested. The plasmid J101-sfgfp@3K3 with green fluorescent protein gene expression in the laboratory was transformed into the tested E. coli through bacteria to form transformation engineering bacteria. Different concentrations of dilutions were performed and the bacteria were spotted on LB and LB+kanamycin solid plates. The transformation efficiency was calculated based on the number of single colonies.

[0018] Secondly, the subculture speed of the strain and the stability of the plasmid during the subculture process were detected. The strain that had been transferred with the J101-sfgfp@3K3 plasmid was selected and the OD was detected by an ELISA instrument. 600 and fluorescence expression to calculate the subculture rate and the average fluorescence of bacteria, to determine whether the strain has an advantage in subculture and whether the plasmid can exist stably for a long time.

[0019] The invention also provides the use of the mouse-derived Escherichia coli in plasmid transformation.

[0020] The present invention also provides the use of the mouse-derived Escherichia coli in mouse intestinal model microorganisms.

[0021] Beneficial effects: The above method steps of the present invention comprehensively utilize the advantages of the green fluorescent protein gene and mouse-derived E. coli, and screen the mouse-derived E. coli with the highest efficiency in all aspects through MEGA evolutionary tree, transformation efficiency and plasmid passage stability experiments. Since it is isolated from the mouse intestine, it can theoretically be colonized in the mouse intestine and used for mouse intestinal related experimental research. Compared with the model E. coli commonly used in mouse intestinal research in existing articles, the strain of the present invention has high transformation efficiency and high plasmid stability, and therefore has more advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0023] Figure 1It is a MEGA evolutionary tree diagram of the mouse-derived isolated bacteria of the present invention and the studied Escherichia coli 16S gene sequence.

[0024] Figure 2 The figure is a comparison of the plasmid transformation effects of the mouse-derived isolated bacteria of the present invention, E. coli Nissle 1917 and E. coli MG1655.

[0025] Figure 3 The figure is a comparison of the plasmid stability of the mouse-derived isolate of the present invention, E. coli Nissle 1917 and E. coli MG1655 during the passage process. DETAILED DESCRIPTION

[0026] Material:

[0027] C57BL / 6 female mice aged 22 weeks were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.

[0028] 2× Max Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0029] Kanamycin was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0030] Tryptone was purchased from Shanghai Biotechnology Co., Ltd.

[0031] Yeast extract was purchased from Shanghai Shenggong Co., Ltd.

[0032] Sodium chloride was purchased from Xilong Scientific Co., Ltd.

[0033] Agar powder was purchased from Lanjieke Technology Co., Ltd.

[0034] Escherichia coli chromogenic culture medium was purchased from Qingdao Haibo Biological Company

[0035] Sterile PBS buffer was purchased from Sewell Biotechnology

[0036] Greiner 96uClear flat bottom ELISA plate was purchased from Guangzhou Dingguo Biotechnology Co., Ltd.

[0037] LB solid culture medium: 10g tryptone, 5g yeast extract, 5g sodium chloride, 20g agar powder, add ultrapure water to 1L; sterilize in an autoclave at 121℃ / 20min, then cool down and pour into the plate.

[0038] LB + Kanamycin solid medium: 10g tryptone, 5g yeast extract, 5g sodium chloride, 20g agar powder, add ultrapure water to 1L, sterilize and cool to 45℃, add 50mg / mL kanamycin solution at a ratio of 1:1000.

[0039] TB liquid medium: 12 g tryptone, 24 g yeast extract, 17 mM KH2PO4, 72 mM K2HPO4, 0.4% glycerol, and ultrapure water to 1 L;

[0040] instrument:

[0041] The microplate reader was purchased from BioTek Instruments model Synergy H1

[0042] High-speed refrigerated centrifuge purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd. Model H1750R

[0043] Electronic balance purchased from Youke Instruments Model JA2003N

[0044] The PCR instrument was purchased from Bio-Rad model T100 TM Thermal Cycler

[0045] The electric constant temperature water bath was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd. Model HWS-24

[0046] The autoclave was purchased from Shanghai Boneng Instrument Co., Ltd. Model CT65A

[0047] Ultrapure water purifier purchased from Guangzhou Dongrui Technology Co., Ltd. Model Directangle-

[0048] Example 1 Screening of mouse-derived Escherichia coli and establishment of MEGA evolutionary tree

[0049] (1) Collection of mouse feces

[0050] C57BL / 6 female mice were dissected at 22 weeks of age, and a pellet of colorectal contents was taken and placed in a centrifuge tube, followed by the following treatments.

[0051] (2) Isolation and identification of mouse-derived Escherichia coli

[0052] Dissolve the collected fresh mouse colorectal contents in 1 mL of sterile PBS and vortex to make a stock solution. Pipette 10 μL of the stock solution and add sterile PBS buffer to 1 mL to obtain 10 -1 dilution, and so on to get 10 -2 -10 -6 diluent. Centrifuge at 12000rpm / min for 1min, discard 900μL of supernatant, apply the remaining on LB solid medium, and grow overnight in a 37℃ incubator. Pick a single colony with neat round edges, smooth surface, translucent, and small protrusions resembling E. coli. After streaking on LB solid medium for 2 times, pick a single colony and streak on E. coli colorimetric solid medium to determine the genus. If the single colony is blue, it is identified as E. coli.

[0053] (3) Construction of MEGA evolutionary tree

[0054] A single colony of Escherichia coli was picked, and the primers in Table 1 and the PCR reaction system in Table 2 were used to perform amplification in Table 3, and a 16S gene fragment of about 1200 bp was amplified by PCR.

[0055] Table 1 PCR primers for the identification of isolated mouse Escherichia coli

[0056]

[0057] Table 2 Amplification process using PCR reaction system (50 μL)

[0058]

[0059] Table 3 PCR amplification process

[0060]

[0061]

[0062] After the reaction was terminated, the purified strain was recovered by 1% agarose gel electrophoresis and subjected to Sanger sequencing (Shanghai Sangon Biotechnology Co., Ltd.). The isolated bacteria were further identified as Escherichia coli by the 16S gene fragment. The bacteria was named Escherichia coli G21229 and preserved in Guangdong Provincial Microbiological Culture Collection Center with a preservation number of GDMCC.No65288 and a preservation date of October 18, 2024.

[0063] The Neighbor-Joining (NJ) method of MEGA software was used to compare the sequences of the isolated mouse E. coli with those of the E. coli commonly used to study the mouse intestine, and differences were found in the sequences, such as Figure 1 shown.

[0064] Example 2: Transformation efficiency of plasmids in mouse Escherichia coli and self-made competent cells of common Escherichia coli

[0065] The existing plasmid J101-sfgfp@3K3 in the laboratory was transformed into each homemade competent cell of E. coli.

[0066] Among them, the competent state preparation adopts the CaCl2 method. A small amount of the preserved strain is streaked on the LB plate and cultured at 37°C. A single colony on the plate is picked to 5mL LB, and cultured at 37°C 180rpm with shaking for 17h. The overnight culture bacteria are diluted 400 times to fresh 200mL LB, and cultured at 37°C 180rpm with shaking until OD600 = 0.3-0.4. The culture is transferred to a 50mL centrifuge tube and immediately placed on ice for 20min. The centrifuge tube is placed in a centrifuge precooled at 4°C, and centrifuged at 3000g for 8min. Discard the supernatant, add 10 mL of pre-cooled 50 mM CaCl2, gently resuspend on ice, repeat the previous centrifugation step to collect the cells, discard the supernatant again, add 10 mL of pre-cooled 50 mM CaCl2, gently resuspend on ice, place on ice for 30 min, centrifuge again at 3000 g for 6 min, discard the supernatant again, add 2 mL of 50 mM CaCl2 and 15% glycerol to each 50 mL centrifuge tube, gently resuspend, place on ice for 2 h, then aliquot into 1.5 mL centrifuge tubes, quick-freeze with liquid nitrogen, and store at -80°C.

[0067] Among them, the transformation of the plasmid adopts the heat shock transformation method, and the plasmid J101-sfgfp@3K3 is transferred into 100μL E. coli competent cells with a final concentration of 1μg / mL. According to the conventional transformation steps, the plasmid is added to the competent cells and placed on ice for 30min, heat-shocked at 42℃ for 90s, immediately placed on ice for 3min, and 900μL fresh LB is added for recovery and culture at 37℃ for 1h. The transformation product is gradient diluted, and 3μL of each is spotted on LB and LB+kanamycin solid plates, repeated three times, and inverted cultured at 37℃ for 17h. Calculated according to the number of colonies at the same concentration, the transformation efficiency % = (number of single colonies on LB+kanamycin solid plate) / (number of single colonies on LB solid plate × dilution factor) × 100%.

[0068] The transformation efficiency of E. coli obtained by calculation is shown in Table 4. The transformation efficiency of mouse E. coli G21229 is 13 times higher than that of the commonly used model E. coli Nissle 1917.

[0069] Table 4 Transformation efficiency of mouse-derived Escherichia coli and common model Escherichia coli

[0070]

[0071] Example 3: Comparison of the passage speed and plasmid stability between mouse Escherichia coli and common model Escherichia coli

[0072] From the E. coli containing plasmid J101-sfgfp@3K3 obtained in Example 2, a single colony was picked and mixed evenly with 70 μL TB liquid culture medium, 170 μL TB was added to the ELISA plate, and then 30 μL of bacterial solution was drawn and mixed evenly with 170 μL TB, and cultured in an ELISA instrument at 37°C and 360 rpm for 17 h as the first generation, and each bacterium was repeated three times. Then 198 μL of fresh TB was added to a new ELISA plate, 2 μL of the first generation bacterial solution was drawn and mixed evenly with 198 μL of fresh TB, and subcultured at 37°C and 360 rpm, and OD was measured at a wavelength of 600 every 30 minutes. 600 The fluorescence intensity was measured with an excitation light of 480, an emission light of 515, and a gain of 50. Then, the cells were diluted 1:100 into 200 μL of fresh TB liquid culture medium every 12 hours for subculture and the OD was measured. 600 A total of 15 dilutions were performed, with a total of 60-70 passages.

[0073] The number of passages of E. coli = log2(A t / A0), A0 is the OD of the cell culture after dilution 600 Value, A t is the OD of the diluted culture after 12 hours of incubation 600 The value was used to calculate the number of passages of different E. coli after 15 dilutions.

[0074] The results in Table 5 were obtained by calculation, in which the mouse-isolated E. coli G21229 had more passage times than the commonly used model E. coli MG1655.

[0075] Table 5 Comparison of the number of passages of Escherichia coli isolated from mice and commonly used model Escherichia coli

[0076]

[0077] Note: The dilution was performed once every 12 hours, for a total of 15 times, so the number of passages calculated in Table 5 is 180 hours.

[0078] The plasmid stability of E. coli is determined by the expression of fluorescence to determine whether the plasmid can exist stably in E. coli for a long time. If the average fluorescence of the bacteria decreases during the passage process, it means that the plasmid has failed and the plasmid stability is poor. The fluorescence value at the 12th hour of each dilution is normalized and compared between different E. coli. Figure 3 The results showed that the Escherichia coli G21229 isolated from mice had the lowest plasmid failure rate and the strongest stability during the subculture process.

[0079] Finally, based on the above three experiments, the mouse-derived E. coli G21229 of the present invention, because it is isolated from the mouse intestine, can theoretically colonize in the mouse intestine for a long time, and its plasmid transformation ability and plasmid stability are better than those of the E. coli commonly used in mouse intestinal research, and has stronger genetic manipulation capabilities.

[0080] Figure 1 : MEGA phylogenetic tree of the 16S gene sequences of the mouse-derived isolated bacteria E. coli G21229 of the present invention and the commonly used E. coli E. coli MG1655, E. coli Nissle 1917 and other closely related species. The 16S gene sequences of other species except E. coli G21229 are from the NCBI database

[0081] Figure 2 :Comparison of plasmid transformation effects of the mouse-derived isolated bacteria E.coli G21229 of the present invention and the commonly used model Escherichia coli E.coli MG1655 and E.coli Nissle 1917. (a) Area map of bacterial solution spotting plate after dilution at different concentrations after bacterial transformation; (b) Colony distribution number of E.coli MG1655, E.coli Nissle 1917 and E.coli G21229 on LB+kanamycin solid medium; (c) Colony distribution number of E.coli MG1655, E.coli Nissle 1917 and E.coli G21229 on LB solid medium; (d) Dilution multiples and single colony statistics and transformation efficiency table of E.coli MG1655, E.coli Nissle 1917 and E.coli G21229 on LB+kanamycin solid medium and LB solid medium.

[0082] Figure 3 : A comparison of the plasmid failure rates of the mouse-derived isolated bacteria E. coli G21229 and the commonly used E. coli MG1655 and E. coli Nissle 1917 in mouse intestinal research during the passage process. The plasmid failure rate of E. coli G21229 is about 1 / 2 of the failure rate of the commonly used model E. coli E. coli MG1655 and E. coli Nissle 1917 at 180h of passage.

[0083] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A strain of mouse-derived Escherichia coli, characterized in that: The mouse-derived Escherichia coli is Escherichia coli G21229, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC.No: 65288 and a deposit date of October 18, 2024.

2. The mouse-derived Escherichia coli according to claim 1, characterized in that The plasmid transformation efficiency of the mouse Escherichia coli reached 3.60×10 -5 , the number of passages reached 69 times.

3. The mouse-derived Escherichia coli according to claim 1, characterized in that The plasmid expiration rate during subculture was lower than that of E.coli MG1655 and E.coli Nissle 1917.

4. The method for culturing mouse-derived Escherichia coli according to any one of claims 1, 2 or 3, characterized in that: The following steps are involved: A small amount of the preserved strain Escherichia coli G21229 was streaked onto an LB solid plate at a culture temperature of 37°C. After culture at 37°C, white translucent round smooth colonies were formed on the LB solid plate.

5. Use of the murine Escherichia coli as described in any one of claims 1, 2 or 3 in plasmid transformation.

6. Use of the mouse-derived Escherichia coli as described in any one of claims 1, 2 or 3 in mouse intestinal model microorganisms.

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