CN111826334A - A kind of super-long Escherichia coli and its preparation method and application - Google Patents

Abstract

The invention discloses an ultralong escherichia coli, and a preparation method and application thereof. The ultra-long escherichia coli obtained by the method ensures the strength of sludge flocs in the activated sludge, improves the precipitation performance of the sludge and influences the purification efficiency of effluent, so that the method can be widely applied to the treatment of wastewater by an activated sludge method; the increase of the size of the bacteria has important influence on the detection of the bacteria, and larger bacteria have more surface antigens and binding sites, so the detection sensitivity is improved, therefore, the invention can be widely applied to the detection of the bacteria; the ultra-long escherichia coli has stronger capability of taking food, stronger capability of resisting external adverse risks and phagocytosis and higher survival rate than normal cells, so that the method can be widely applied to scientific research with requirements on the survival rate of bacteria; the ultra-long escherichia coli generated by the method is longer, and has practical significance and practical operability by considering the combined action of multiple environmental factors.

Description

A kind of super-long Escherichia coli and its preparation method and application

technical field

The invention belongs to the field of biotechnology, and relates to a super-long Escherichia coli and a preparation method and application thereof.

Background technique

In many fields of scientific research, bacterial size has a significant impact on bacterial detection, such as oxidative stress detection, motility and chemotaxis studies, antibiotic susceptibility, single-cell detection, and overall imaging analysis. Detection sensitivity is improved because larger bacteria have more surface antigens and binding sites. In addition, the formation of ultra-long E. coli plays an important role in the treatment of sewage by activated sludge process. In activated sludge, ultra-long E. coli can act as a skeleton to ensure the strength of the sludge flocs, improve the sedimentation performance of the sludge, and affect the Purification efficiency of effluent. Furthermore, superlong Escherichia coli is the product of cell division without cleavage caused by fission phase defects. It has stronger resistance to phagocytosis and resistance to adverse environments. It has a stronger ability to ingest food than normal bacteria, and its survival rate is higher than normal. Cells are high.

Adverse environmental stresses such as SOS reaction, DNA damage, high pressure, and dehydration have been reported to lead to the formation of ultralong E. coli. Studies have shown that antibiotics can promote the growth of E. coli, such as cephalexin. There are two mechanisms by which cephalexin induces the formation of ultra-long E. coli. The first is to inhibit the formation of cross-links of the peptidoglycan layer and the synthesis of the cell wall by binding to penicillin-binding proteins (PBPs) on the cell wall, resulting in the growth of the cell wall. Does not divide; a second possible mechanism is the SOS response caused by the antibiotic effect of cephalexin, resulting in incomplete cell membranes and cell walls. The concentration of cephalexin also affects the elongation effect of E. coli. When the concentration of cephalexin is lower than the minimum inhibitory concentration, ultra-long E. coli can be formed, but when the concentration is higher, it will lead to bacterial inactivation. Although antibiotics can induce the formation of ultra-long Escherichia coli, this only takes into account the unilateral effect of antibiotics, and only prolonging the length of Escherichia coli with antibiotics cannot meet the needs of current scientific research.

UV irradiation is a physical method to form ultralong E. coli, which activates specific genes and induces SOS response after damaging or inhibiting the replication of intracellular DNA through continuous UV irradiation. The SOS reaction can inhibit bacterial division, allowing bacteria to only grow without dividing, thereby forming superlong E. coli. The type of UV light, light intensity, time and frequency will affect the formation of ultra-long E. coli. The ultra-long E. coli produced by UV irradiation has a low survival rate due to DNA damage, and has poor heritability. Once the damaged DNA is repaired, the cell division ability will slowly recover.

Therefore, the method for preparing ultra-long Escherichia coli of the present invention has great value for bacterial detection, wastewater treatment and other scientific researches that require bacterial survival rate.

SUMMARY OF THE INVENTION

In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing ultra-long Escherichia coli.

Another object of the present invention is to provide ultralong Escherichia coli prepared by the above preparation method

Another object of the present invention is to provide the application of the above-mentioned ultra-long Escherichia coli.

The object of the present invention is achieved through the following technical solutions: a preparation method of ultra-long Escherichia coli, comprising the steps:

(1) inoculate Escherichia coli into the medium, cultivate, dilute, and cultivate again;

(2) inoculating the Escherichia coli after recultivation in step (1) into a medium containing surfactant and cephalexin, and culturing to obtain ultralong Escherichia coli.

Escherichia coli described in the step (1) is made to have resistance to other antibiotics except cephalexin by the method of genetic engineering before use, or directly use Escherichia coli with other antibiotic resistance, and in the culturing process. Add the appropriate amount of this antibiotic.

The Escherichia coli described in step (1) is preferably one of E.coli TOP 10, E.coli pD1B10, E.coli BL21, and E.coli MG1655; more preferably E.coli TOP 10.

The medium described in step (1) is LB medium with a pH value of 7.0.

The formulation of the LB medium: 10 g/L sodium chloride, 5 g/L yeast extract and 10 g/L tryptone.

The culture described in step (1) is 37° C. for 12 hours until the absorbance value OD=0.5.

The dilution described in step (1) is 1000-fold dilution.

The re-cultivation described in step (1) is 37° C. for 2 h.

The dosage of cephalexin described in the step (2) is 30-120 μg/mL according to its concentration in the culture medium; preferably 60-100 μg/mL; more preferably 60-90 μg/mL.

The surfactants described in step (2) are preferably cationic surfactant CTAB, anionic surfactant SDS and nonionic surfactant Tween.

The nonionic surfactant Tween is preferably Tween-20.

When the surfactant is CTAB, its dosage is 0.0005-0.02% by mass according to its concentration in the culture medium; preferably, it is 0.0005-0.002% by mass according to its concentration in the culture medium; Preferably, it is formulated according to its concentration in the medium as a mass ratio of 0.001%.

When the surfactant is SDS, its dosage is 0.005-0.5% by mass according to its concentration in the medium; preferably, it is 0.1% by mass according to its concentration in the medium.

When the surfactant is Tween-20, its dosage is 0.02-10% by mass according to its concentration in the medium; preferably, it is 1-2% according to its concentration in the medium.

The culturing in step (2) is 37°C for 4-8 hours; preferably 4 hours.

During the culturing and re-cultivation of steps (1) and (2), when a 250 mL conical flask is used for shaking the bacteria, the speed of shaking the bacteria is preferably 60 rpm.

An ultra-long Escherichia coli is prepared by the above preparation method.

The application of the above-mentioned ultra-long Escherichia coli in the treatment of wastewater by an activated sludge process.

Application of the above-mentioned ultra-long Escherichia coli in bacterial detection.

Compared with the prior art, the present invention has the following advantages and effects:

1. The ultra-long Escherichia coli prepared by the method of the present invention can act as a skeleton in the activated sludge, ensure the strength of the sludge flocs, improve the sedimentation performance of the sludge, and affect the purification efficiency of the effluent. Therefore, the present invention can be widely used in the treatment of wastewater by the activated sludge method.

2. The increase in bacterial size has important effects on bacterial detection, such as oxidative stress detection, motility and chemotaxis studies, antibiotic susceptibility, single-cell detection and overall imaging analysis. Larger bacteria have more surface antigens and binding sites, which increases detection sensitivity. Therefore, the ultra-long Escherichia coli obtained by the method of the present invention can be widely used in bacterial detection.

3. Ultra-long E. coli has a stronger ability to ingest food than normal bacteria, and also has a stronger ability to resist external dangers and phagocytosis. The survival rate of ultra-long E. coli during bacterial culture is higher than that of normal cells. . Therefore, the ultra-long Escherichia coli obtained by the method of the present invention can be widely used in scientific research requiring bacterial survival rate.

4. Compared with the super-long Escherichia coli produced only with antibiotics, the super-long Escherichia coli produced by the combined action of surfactants and antibiotics is longer, and the longest is about 3 times that when cephalexin acts alone; at the same time, the technology of the present invention considers When it comes to the joint action of multiple environmental factors, it is more realistic and practical than considering the single action of cephalexin.

5. The UV treatment method will cause DNA damage in Escherichia coli, resulting in gene mutation, while this method will not cause DNA damage in principle and thus reduce the probability of gene mutation. Compared with the ultra-long E. coli produced by UV technology, the ultra-long E. coli produced by the combination of surfactants and antibiotics has a normal DNA distribution state and a higher survival rate.

Description of drawings

FIG. 1 is an experimental flow chart of Examples 1-3.

Fig. 2 The length statistics of the 10 longest E. coli obtained by treating E. coli with different concentrations of CTAB in combination with 90 μg/mL cephalexin.

Fig. 3 is a graph of the length statistics of the 10 longest E. coli obtained by treating E. coli with different concentrations of SDS and 90 μg/mL cephalexin.

Figure 4 is a graph showing the length statistics of the 10 longest E. coli obtained by treating E. coli with different concentrations of Tween-20 in combination with 90 μg/mL cephalexin.

Fig. 5 is the distribution map of DNA in superlong E. coli; wherein, a is E. coli DNA treated with cephalexin and surfactant; b is an enlarged image of the area within the white square in a; the scale bar length of a is 3 μm; b The scale bar length is 5 μm.

Figure 6 is the distribution diagram of the cell membrane in superlong Escherichia coli; wherein, a is the E. coli cell membrane treated with cephalexin and surfactant; b is the enlarged image of the area inside the white square in a; the scale bar length of a is 3 μm; b The scale bar length is 5 μm.

Figure 7 is a graph of Zeta potential statistics of untreated, cephalexin treated, cephalexin and surfactant (CTAB, SDS and Tween-20) treated E. coli.

Figure 8 is a graph showing the length statistics of ultra-long Escherichia coli obtained after treatment with different concentrations of cephalexin; wherein, the length of bacteria is shown as black dots, and the average value of the standard deviation of each group of data is shown by a horizontal line.

Fig. 9 is a microscope view of Escherichia coli cultured for different time with cephalexin at a concentration of 90 μg/mL; wherein, a is cultured for 0 h; b is cultured for 4 hours; c is cultured for 8 hours.

Figure 10 is an electron microscope image of Escherichia coli treated with cephalexin; the scale bar is 60 μm.

Figure 11 is a statistical graph of the concentration of cephalexin required for the preparation of ultralong Escherichia coli by different Escherichia coli strains.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

The reagents and raw materials not marked in the present invention are all obtained from the market.

Example 1 Production of ultralong Escherichia coli by CTAB

CTAB is a white or light yellow solid substance, easily soluble in isopropanol, soluble in water, and has good biodegradation and bactericidal properties. The CTAB solution was prepared with water, and its concentration in the medium was 0.0005% (w/ w), 0.001% (w/w), 0.002% (w/w), 0.005% (w/w), 0.01% (w/w) and 0.02% (w/w).

①Inoculate E.coli TOP 10 into LB medium containing 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone, pH 7.0, and aerobic at 37℃, 60rpm Under the condition of overnight culture for 12h (using a 250mL conical flask, the same below).

② Dilute the bacterial liquid cultured for 12 hours and the absorbance value is 0.5 (OD=0.5) by 1000 times with the medium, inoculate the diluted E.

③ Inoculate the E. coli after re-cultivation in ② to 0.0005% (w/w) CTAB+90 μg/mL cephalexin, 0.001% (w/w) CTAB+90 μg/mL cephalexin, 0.002% (w/w) CTAB + 90 μg/mL cephalexin, 0.005% (w/w) CTAB + 90 μg/mL cephalexin, 0.01% (w/w) CTAB + 90 μg/mL cephalexin and 0.02% (w/w) CTAB + 90 μg/mL In the medium of cephalexin, aerobic cultivation was performed at 37°C and 60 rpm for 4 h, and the bacterial lengths were measured after the cultivation. Control settings: negative control without addition of cephalexin and CTAB (WT), positive control with addition of 90 μg/mL cephalexin (Cex). The experimental flow chart is shown in Figure 1.

Example 2 SDS production of ultra-long Escherichia coli

SDS is a white or light yellow micro-sticky substance that is easily soluble in water and has good emulsifying and decontamination properties. Since the maximum concentration of SDS in water is limited by its solubility, the concentrations used are not very high. The solutions prepared with water have concentrations of 0.005% (w/w) and 0.02% (w/w) in the medium, respectively. ), 0.05% (w/w), 0.1% (w/w), 0.2% (w/w) and 0.5% (w/w).

①Escherichia coli was inoculated into LB medium containing 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone, pH 7.0, and cultured overnight at 37°C and 60rpm under aerobic conditions for 12h (Use a 250mL conical flask, the same below).

② Dilute the bacterial liquid cultured for 12 hours and the absorbance value is 0.5 (OD=0.5) by 1000 times with the medium, inoculate the diluted E.

③ Inoculate the E. coli after re-cultivation in ② to 0.005% (w/w) SDS+90 μg/mL cephalexin, 0.02% (w/w) SDS+90 μg/mL cephalexin, 0.05% (w/w) SDS + 90 μg/mL cephalexin, 0.1% (w/w) SDS + 90 μg/mL cephalexin, 0.2% (w/w) SDS + 90 μg/mL cephalexin and 0.5% (w/w) SDS + 90 μg/mL In the medium of cephalexin, aerobic cultivation was performed at 37°C and 60 rpm for 4 h, and the bacterial lengths were measured after the cultivation. Control settings: negative control without addition of cephalexin and SDS (WT), positive control with addition of 90 μg/mL cephalexin (Cex).

Example 3 Tween-20 manufactures ultralong Escherichia coli

Tween-20 has strong hydrophilic properties because its molecular formula contains many hydrophilic groups. It is often used as an oil-in-water (O/W) emulsifier and can be used in combination with other emulsifiers to enhance the stability of the emulsifier. . Due to the low toxicity of Tween-20, the concentration of Tween-20 is higher than that of CTAB and SDS (prepared in water). w/w), 2% (w/w) and 10% (w/w) six concentrations.

①Escherichia coli was inoculated into LB medium containing 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone, pH 7.0, and cultured overnight at 37°C and 60rpm under aerobic conditions for 12h (Use a 250mL conical flask, the same below).

② Dilute the bacterial liquid cultured for 12 hours and the absorbance value is 0.5 (OD=0.5) by 1000 times with the medium, inoculate the diluted E.

③ Inoculate the Escherichia coli after re-cultivation in ② to 0.02% (w/w) Tween-20+90 μg/mL cephalexin, 0.1% (w/w) Tween-20+90 μg/mL cephalexin, 0.2% (w/w) Tween-20+90 μg/mL cephalexin, respectively. w/w) Tween-20 + 90 μg/mL cephalexin, 1% (w/w) Tween-20 + 90 μg/mL cephalexin, 2% (w/w) Tween-20 + 90 μg/mL cephalexin and 10% (w/w) Tween-20+90μg/mL cephalexin medium, 37 ℃, 60rpm aerobic culture for 4h, the length of bacteria were measured after the end of the culture. Control settings: negative control without addition of cephalexin and Tween-20 (WT), positive control with addition of 90 μg/mL cephalexin (Cex).

Example 4 Characterization of ultralong Escherichia coli

(1) Calculation of the average length of ultra-long Escherichia coli

According to the following three hypotheses: 1) the surfactant does not change the charge on the surface of E. coli, 2) the charge is uniformly distributed along the E. coli, and 3) the E. coli is in the shape of a rod-shaped column, the calculation of cephalexin, cephalexin and surfactant-treated The average length of the E. coli samples (Examples 1-3).

Calculation method:

The relationship between electrophoretic mobility and E. coli charge: Q=6πηrμ (1)

The relationship between zeta potential z and electrophoretic mobility is:

The relationship between the zeta potential z and the charge of Escherichia coli is obtained by combining formula (1) and (2): Q=4πrεzf(ka)(3)

Assuming that E. coli is a cylinder of diameter d, length L, and ratio n=L/d, the diffusion coefficient D is:

According to the Einstein-Stokes relationship:

Combining equations (4) and (5), we get:

Assuming that the charge is uniformly distributed along E. coli, we get: Q=Adn (7)

Combine formulas (3) (6) (7) to get:

According to formula (8), the relationship between the zeta potential z and the length of Escherichia coli is finally obtained:

where Q is the net charge of E. coli, η is the viscosity of water, r is the hydrodynamic radius of E. coli, and μ is the electrophoretic mobility. ε is the permittivity of water and f(ka) is the Henry function (according to the Smoluchowski approximation, it is 1.5). k is the Boltzmann constant and T is the absolute temperature.

It can be seen from the final formula that the zeta potential z is related to n, and n=L/d, so in fact, the zeta potential z on the E. coli cell surface is positively correlated with the length of the bacteria, that is, the higher the zeta potential on the cell surface, the longer the bacteria.

The length of Escherichia coli prepared in Example 1 is shown in Figure 2. It can be seen from the figure that the average length ratio of the Escherichia coli produced in combination with CTAB and cephalexin at a concentration of 0.005% (w/w) and 0.02% (w/w) The E. coli produced by cephalexin alone was slightly shorter, and the prolongation effect of 0.005% (w/w) CTAB was the least ideal. CTAB at concentrations of 0.0005% (w/w), 0.001% (w/w), 0.002% (w/w) and 0.01% (w/w) all enhanced the effect of cephalexin in prolonging E. coli, with 0.001% ( w/w) the enhancement effect was the best, about 1.5 times that of cephalexin (Cex) alone.

The length of Escherichia coli prepared in Example 2 is shown in Figure 3. It can be seen from the figure that five different concentrations of SDS have enhanced the effect of cephalexin in extending Escherichia coli, and the enhancement effect of 0.5% (w/w) is the least obvious. , 0.1% (w/w) SDS had the best enhancement effect, which was about 2 times that of cephalexin alone. At the same time, it can also be seen that the prolongation effect of 0.05% (w/w) and 0.2% (w/w) is equivalent to about 1.8 times that of cephalexin alone, and the prolongation effect of 0.005% (w/w) is higher than that of 0.02%. (w/w) more obvious.

The length of Escherichia coli prepared in Example 3 is shown in Figure 4. As can be seen from the figure, different concentrations of Tween-20 have enhancement effects on the extension of Escherichia coli by cephalexin, and there are significant differences in the extension effects. 0.02% (w/w), 0.1% and 0.2% (w/w) Tween-20 had similar effects on enhancing the elongation of E. coli by cephalexin, and the length distributions were all uniform. Both 1% (w/w) and 2% (w/w) Tween-20 have significant elongation effects on E. coli, and 1% (w/w) Tween-20 has the best elongation effect, which is about cephalexin 3 times when acting alone.

(2) Membrane integrity and DNA distribution of ultralong Escherichia coli

Surfactants are cytotoxic, such as inducing lysis of Gram-positive and Gram-negative bacteria, inhibiting bacterial growth, and altering cell wall structure and porosity. At the same time, cephalexin as an antibiotic has a certain bacteriostatic effect. Therefore, it is necessary to determine whether the bacteria treated with surfactant combined with cephalexin and the bacteria treated with cephalexin have normal cell activity.

①Collect the Escherichia coli bacteria that was normally cultured for 4 hours under aerobic conditions at 37°C and 60rpm.

②Collect Escherichia coli exposed to 90 μg/mL cephalexin for 4 h under aerobic conditions at 37°C and 60 rpm (with cephalexin added to LB medium).

③ Collect the 0.001% (W/W) CTAB+90 μg/mL cephalexin, 0.1% (W/W) SDS+90 μg/mL cephalexin and 1% (W/W) Tween-20+90 μg/mL cephalexin respectively Co-exposed Escherichia coli (surfactant and cephalexin in LB medium) for 4 h.

④ Take 500 μL of bacterial samples in ①②③ and stain with 200 μL of DAPI for 5 min, and then centrifuge at 8000 rpm for 5 min to collect bacterial samples.

⑤ Resuspend the bacterial particles in ④ in 200 μL of FM4-64 solution, incubate at 0°C for 1 minute, and then centrifuge the suspension at 8000 rpm for 5 minutes to collect the bacterial particles and resuspend them in distilled water.

⑥The bacterial cell membrane and DNA in ⑤ were observed by fluorescence microscope respectively.

The distribution of DNA is shown in Figure 5. The DNA of ultra-long E. coli produced by the combined action of surfactants and antibiotics is longer than that of normal cells and ultra-long bacteria produced by cephalexin, and is normally distributed in cells. E. coli was evenly distributed longitudinally.

The distribution of the cell membrane is shown in Figure 6. The cell membrane of the ultra-long E. coli produced by the combined action of surfactants and antibiotics is longer than that of normal cells and the ultra-long bacteria produced by the action of cephalexin, and it is continuously and evenly distributed on the surface of E. coli , the surface morphology is normal.

Therefore, bacteria treated with surfactant in combination with cephalexin have normal cellular activity.

(3) Interaction between surfactants and cells

Surfactants can interact with cell membranes, promote the transmembrane transport of organic matter, and improve the permeability of cell membranes. Surfactants are structurally similar to lipids on cell membranes, so it is likely that surfactants interact with E. coli through bulk insertion into the cell membrane, a hypothesis we tested with zeta potential measurements. The zeta potential of Escherichia coli is proportional to the charge. When a large amount of surfactant is inserted or bound to the cell surface (including the cell membrane and cell wall), the zeta potential will change. For SDS and CTAB with different charges, the zeta potential generated by the zeta potential will change. should be different.

①Collect the Escherichia coli bacteria that was normally cultured for 4 hours under aerobic conditions at 37°C and 60rpm.

② Collect Escherichia coli exposed to 90 μg/mL cephalexin for 4 h (with cephalexin added to LB medium) under aerobic conditions at 37°C and 60 rpm.

③ Collected under aerobic conditions at 37°C and 60 rpm, respectively, after 0.001% (W/W) CTAB+90 μg/mL cephalexin, 0.1% (W/W) SDS+90 μg/mL cephalexin and 1% (W/W) Tween-20+90μg/mL cephalexin was exposed to Escherichia coli for 4h (surfactant and cephalexin were added to LB medium).

④ Centrifuge the Escherichia coli samples in ② and ③ at 8000 rpm for 5 min respectively. After the centrifugation, collect bacterial particles and resuspend them in pure water.

⑤ The cell membrane surface potentials of E. coli in ① and ④ were measured with Zeta-PALS (ZETAPALS/BI-200SM, Brookhaven, USA) respectively, and the measurements were carried out three times under the same experimental conditions.

The results are shown in Figure 7. Whether it is E. coli treated with cephalexin alone or E. coli treated with surfactant and cephalexin in combination, the Zeta potential of the cell membrane surface is higher than that of untreated E. coli. Low. At the same time, the zeta potential of E. coli treated with cephalexin alone changed significantly. However, the zeta potentials of the E. coli samples treated with surfactant and cephalexin were almost identical to those of the E. coli samples treated with cephalexin alone.

The above results indicated that the surfactant did not insert or bind to the cell surface and did not change the normal function of E. coli.

Comparative Example 1 Traditional technology to manufacture ultra-long Escherichia coli

①Escherichia coli was inoculated into LB medium containing 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone, pH 7.0, and cultured overnight at 37°C and 60rpm under aerobic conditions for 12h (Use a 250mL conical flask, the same below).

② Dilute the bacterial liquid cultured for 12 hours and the absorbance value is 0.5 (OD=0.5) by 1000 times with the medium, inoculate the diluted E.

③ Inoculate the bacteria in ② into the LB medium of 80 μg/mL cephalexin, 90 μg/mL cephalexin and 100 μg/mL cephalexin respectively, and incubate them aerobic at 37°C and 60 rpm for 4 hours, and measure the bacterial length after the culture.

④ The bacteria in ② were inoculated into 80 μg/mL cephalexin, 90 μg/mL cephalexin and 100 μg/mL cephalexin LB medium respectively, and aerobic culture was carried out at 37°C and 60 rpm for 8 h, and the bacterial length was measured after the culture was completed.

⑤ Fifty strains of Escherichia coli treated with different concentrations of cephalexin in ③ and ④ were randomly selected from each group, and the length was measured by electron microscope. The length calculation method is the same as the step (1) of Embodiment 4.

The results are shown in Figures 8 and 9, the optimal treatment concentration of cephalexin is 90 μg/mL, and the optimal treatment time is 4h. Due to the cytotoxicity of cephalexin, the number of cells decreased after culture. Excessive incubation time (8 hours) resulted in depletion of cephalexin and a decrease in concentration, resulting in the disappearance of the prolonged results (Fig. 9c). It shows that the concentration of cephalexin is the key to obtaining ultra-long E. coli, which needs to be controlled by precise incubation time, that is, as the incubation time prolongs, the E. coli gradually increases, and the amount of cephalexin in it gradually decreases. During the first four hours, the amount of cephalexin was still sublethal, as evidenced by the continued growth of E. coli. After four hours, the E. coli gradually recovered as the amount of cephalexin decreased beyond the effective concentration. Strict control of the decay time of cephalexin can maximize the acquisition of ultra-long E. coli. In addition, we carried out electron microscope measurement on the ultra-long E. coli (obtained in step 3) prepared by the traditional method. The measurement results found that the ultra-long E. coli was a long filament with a width of about 1 micrometer, and its length was nearly a hundred times longer than that of ordinary E. coli (Figure 10). At the same time, it was also found that the ultra-long E. coli only accounted for a small part of the total amount of all E. coli, and the vast majority of E. coli were still in an unextended state.

Comparative Example 2

Change the speed of shaking bacteria in steps ①②③ in Example 1 to 0, 30 rpm and 200 rpm respectively, and the other conditions are exactly the same to prepare ultra-long Escherichia coli. Change the container for shaking bacteria in steps ①②③ of Example 1 to 50mL centrifuge tubes, 500mL and 1000mL conical flasks respectively, and the other conditions are exactly the same to prepare ultra-long Escherichia coli.

The results proved that insufficient number of revolutions or static culture (the number of revolutions is 0) when shaking bacteria will lead to slow growth of E. coli, and a sufficient number of E. coli cannot be obtained; on the other hand, if the speed of shaking bacteria is too fast, it will lead to the longest length of E. coli shorten. With the increase of the diameter of the bacteria-containing vessel, the mechanical shearing force produced by the same number of revolutions is very different. At about 60 rpm, the purpose of preparing ultra-long E. coli can be achieved only by shaking the bacteria in a 250 mL conical flask. If a larger diameter bacteria container is used, the number of revolutions should be reduced to avoid excessive mechanical collision leading to bacterial death or rupture of super-long E. coli; if a smaller diameter bacteria container is used, the number of revolutions should be increased accordingly.

Comparative Example 3

①Escherichia coli (E.coli pD1B10, E.coli BL21, E.coli MG1655 strains) were inoculated into pH 7.0 containing 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone respectively LB medium, and cultured at 37° C. and 60 rpm under aerobic conditions for 12 h overnight (using a 250 mL conical flask, the same below).

② Dilute the bacterial liquid cultured for 12 hours and the absorbance value is 0.5 (OD=0.5) by 1000 times with the medium, inoculate the diluted E.

③ The bacteria in ② were inoculated into the LB medium of cephalexin of 30 μg/mL, 45 μg/mL, 60 μg/mL, 75 μg/mL, 90 μg/mL, 105 μg/mL and 120 μg/mL, respectively, at 37 °C, 60 rpm aerobic After culturing for 4 h, the bacterial length was measured at the end of the culture.

The results are shown in FIG. 11 , the concentrations of cephalexin required for the preparation of ultralong Escherichia coli by different Escherichia coli strains are different.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (10)

1. A preparation method of ultra-long escherichia coli is characterized by comprising the following steps:

(1) inoculating Escherichia coli into culture medium, culturing, diluting, and culturing;

(2) inoculating the escherichia coli re-cultured in the step (1) into a culture medium containing a surfactant and cefalexin, and culturing to obtain the ultralong escherichia coli.

2. The method for producing ultra-long Escherichia coli according to claim 1, wherein:

the escherichia coli in the step (1) is one of E.coli TOP 10, E.coli D1B10, E.coli BL21 and E.coli MG1655;

before use, the escherichia coli in the step (1) is made to have the resistance of other antibiotics except for cefalexin by a genetic engineering method, or the escherichia coli with the resistance of other antibiotics is directly used, and a proper amount of the antibiotics is added in the culture process;

the surfactant in the step (2) is cationic surfactant CTAB, anionic surfactant SDS and nonionic surfactant Tween.

3. The method for producing very long E.coli according to claim 2, wherein:

coli TOP 10;

the nonionic surfactant Tween is Tween-20.

4. The method for producing ultra-long Escherichia coli according to claim 3, wherein:

the dosage of the cefalexin in the step (2) is 30-120 mu g/mL;

when the surfactant is CTAB, the dosage of the surfactant is 0.0005-0.02% of the mass ratio of the surfactant in the culture medium;

when the surfactant is SDS, the dosage of the surfactant is 0.005-0.5 percent of the mass ratio of the surfactant in the culture medium;

when the surfactant is Tween-20, the dosage of the surfactant is 0.02-10% of the mass ratio of the surfactant in the culture medium.

5. The method for producing ultra-long Escherichia coli according to claim 4, wherein:

the dosage of the cefalexin in the step (2) is 60-100 mug/mL;

when the surfactant is CTAB, the dosage of the surfactant is 0.001 percent of the mass ratio of the surfactant in the culture medium;

when the surfactant is SDS, the dosage of the surfactant is 0.1 percent of the mass ratio of the surfactant in the culture medium;

when the surfactant is Tween-20, the dosage of the surfactant is 1-2% of the mass ratio of the surfactant in the culture medium.

6. The method for producing ultra-long Escherichia coli according to claim 1, wherein:

the culture medium in the step (1) is an LB culture medium with the pH value of 7.0;

the LB culture medium comprises the following components in percentage by weight: 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone;

the culture in the step (1) is carried out at 37 ℃ for 12h until the absorbance OD is 0.5;

the dilution in the step (1) is 1000 times;

the re-culture in the step (1) is culture at 37 ℃ for 2 h;

the culture in the step (2) is carried out for 4-8h at 37 ℃;

in the culture and re-culture processes of the steps (1) and (2), when a 250mL conical flask is adopted for bacteria shaking, the speed of bacteria shaking is 60 rpm.

7. The method for producing ultra-long Escherichia coli according to claim 6, wherein: the culture in the step (2) is carried out for 4 hours at 37 ℃.

8. An ultra-long escherichia coli, characterized in that: prepared by the preparation method of any one of claims 1 to 7.

9. Use of the ultra-long escherichia coli according to claim 8 for treatment of wastewater by an activated sludge process.

10. Use of the ultra-long E.coli of claim 8 for bacterial detection.

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