CN111826334A - Ultra-long escherichia coli and preparation method and application thereof - Google Patents
Ultra-long escherichia coli and preparation method and application thereof Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of biology, and relates to ultralong escherichia coli, and a preparation method and application thereof.
Background
In many areas of scientific research, bacterial size has a significant impact on bacterial detection, such as oxidative stress detection, motility and chemotaxis studies, antibiotic sensitivity, single cell detection, and whole body imaging analysis. Since larger bacteria have more surface antigens and binding sites, the detection sensitivity can be improved. In addition, the formation of the ultra-long escherichia coli plays an important role in sewage treatment by an activated sludge method, the ultra-long escherichia coli in the activated sludge can serve as a skeleton, the strength of sludge flocs is ensured, the settling property of the sludge is improved, and the purification efficiency of effluent is influenced. In addition, the ultra-long escherichia coli is a product which is not broken due to cell division caused by the division phase defect, has stronger capabilities of resisting phagocytosis and resisting adverse environment, has stronger capability of taking food than bacteria in a normal form, and has higher survival rate than normal cells.
Adverse environmental stresses such as SOS reactions, DNA damage, high pressure and dehydration have been reported to lead to the formation of very long E.coli. It has been shown that antibiotics promote growth of E.coli, such as cephalexin. There are two mechanisms by which cephalexin is induced to form very long E.coli, the first is to inhibit the formation of cross-links of peptidoglycan layers and the synthesis of cell walls by binding to Penicillin Binding Proteins (PBPs) on the cell walls, resulting in cell wall growth without splitting; the second possible mechanism is the SOS reaction caused by the antibiotic effect of cephalexin, resulting in an incomplete cell membrane and cell wall. The concentration of cephalexin also affects the elongation effect of escherichia coli, and when the concentration of cephalexin is lower than the minimum inhibitory concentration, overlength escherichia coli can be formed, but when the concentration is higher, the bacteria can be inactivated. Although antibiotics can induce the formation of ultra-long escherichia coli, only the unilateral effect of the antibiotics is considered, and the extension of the length of escherichia coli by only the antibiotics cannot meet the requirements of current scientific research.
UV light is a physical method of forming very long E.coli by activating specific genes and inducing SOS response after destroying or inhibiting the replication of DNA in cells by continuous UV irradiation. SOS reaction can inhibit bacterial division, and allow bacteria to grow but not divide, thereby forming ultra-long Escherichia coli. The type of UV light, the intensity, duration and frequency of the light, etc., all affect the formation of ultra-long E.coli. The ultra-long E.coli produced by UV irradiation has low survival rate due to DNA damage, and poor heritability, and the division ability of cells is slowly restored once the damaged DNA is repaired.
Therefore, the method for preparing the ultra-long escherichia coli has great value for bacterial detection, wastewater treatment and other scientific researches with requirements on bacterial survival rate.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a preparation method of ultra-long escherichia coli.
Another object of the present invention is to provide a very long Escherichia coli prepared by the above method
Still another object of the present invention is to provide the use of the above-mentioned ultra-long Escherichia coli.
The purpose of the invention is realized by the following technical scheme: a preparation method of ultra-long Escherichia coli comprises 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.
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 escherichia coli in the step (1) is preferably one of E.coli TOP 10, E.coli pD1B10, E.coli BL21 and E.coli MG 1655; coli TOP 10 is more preferred.
The culture medium in the step (1) is 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 step (1) was carried out at 37 ℃ for 12 hours until the absorbance OD was 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 cefalexin in the step (2) is used in a proportion that the concentration of the cefalexin in a culture medium is 30-120 mug/mL; preferably 60-100 mug/mL; more preferably 60 to 90. mu.g/mL.
The surfactant in the step (2) is preferably cationic surfactant CTAB, anionic surfactant SDS and nonionic surfactant Tween.
The non-ionic surfactant Tween is preferably Tween-20.
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; preferably, the concentration of the compound is 0.0005-0.002% by mass in the culture medium; more preferably, the concentration of the compound in the medium is 0.001% by mass.
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; preferably, the concentration of the compound in the culture medium is 0.1% by mass.
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; preferably 1-2% of the culture medium.
The culture in the step (2) is carried out for 4-8h at 37 ℃; preferably 4 hours.
In the culturing and re-culturing processes in the steps (1) and (2), when a 250mL conical flask is adopted for bacteria shaking, the speed of bacteria shaking is preferably 60 rpm.
An ultralong Escherichia coli is prepared by the above preparation method.
The application of the ultra-long escherichia coli in wastewater treatment by an activated sludge process.
The application of the ultra-long escherichia coli in bacterial detection.
Compared with the prior art, the invention has the following advantages and effects:
1. the ultra-long escherichia coli prepared by the method can serve as a skeleton in the activated sludge, so that the strength of sludge flocs is ensured, the settling property of the sludge is improved, and the purification efficiency of effluent is influenced. Therefore, the invention can be widely applied to the treatment of wastewater by an activated sludge method.
2. The increase in bacterial size has important implications for bacterial detection, such as oxidative stress detection, motility and chemotaxis studies, antibiotic sensitivity, single cell detection, and global imaging analysis. Larger bacteria have more surface antigens and binding sites, which can improve detection sensitivity. Therefore, the ultra-long escherichia coli obtained by the method can be widely applied to bacteria detection.
3. The ultra-long escherichia coli has stronger capability of taking food than bacteria in normal forms, and also has stronger capabilities of resisting external adverse risks and resisting phagocytosis, and the survival rate of the ultra-long escherichia coli in the bacterial culture process is higher than that of normal cells. Therefore, the ultra-long escherichia coli obtained by the method can be widely applied to scientific research with requirements on the survival rate of bacteria.
4. Compared with the ultra-long escherichia coli generated only by using antibiotics, the ultra-long escherichia coli generated by the combined action of the surfactant and the antibiotics is longer, and the longest time is about 3 times that of the long escherichia coli generated by the single action of the cefalexin; meanwhile, the technology of the invention considers the combined action of multiple environmental factors, and has more practical significance and actual operability than the consideration of the single action of cefalexin.
5. The UV treatment method can cause DNA damage of escherichia coli and cause gene mutation, and the method can not cause DNA damage in principle and further reduce the probability of gene mutation. The surfactant combined with antibiotics produced very long E.coli with normal DNA distribution and higher survival rate compared to the very long E.coli produced by UV technology.
Drawings
FIG. 1 is a flow chart of the experiment of examples 1-3.
FIG. 2 is a length histogram of 10 longest E.coli strains obtained by treating E.coli with CTAB at various concentrations in combination with 90. mu.g/mL cephalexin.
FIG. 3 is a length histogram of 10 longest E.coli strains obtained by treating E.coli strains with different concentrations of SDS in combination with 90. mu.g/mL cephalexin.
FIG. 4 is a length statistical plot of the 10 longest E.coli strains resulting from treatment of E.coli with different concentrations of Tween-20 in combination with 90. mu.g/mL cephalexin.
FIG. 5 is a map of DNA distribution in ultra-long E.coli; wherein a is escherichia coli DNA treated by cefalexin and a surfactant; b is the enlarged image of the area in the white square in a; the length of a scale bar is 3 mu m; the scale bar length of b is 5 μm.
FIG. 6 is a distribution diagram of cell membranes in very long E.coli; wherein a is an escherichia coli cell membrane treated by cefalexin and a surfactant; b is the enlarged image of the area in the white square in a; the length of a scale bar is 3 mu m; the scale bar length of b is 5 μm.
FIG. 7 is a Zeta potential statistical plot for untreated, cephalexin treated, cephalexin and surfactant (CTAB, SDS and Tween-20) treated E.coli.
FIG. 8 is a statistical graph of the lengths of very long E.coli cells obtained after treatment with cephalexin at different concentrations; where the length of the bacteria is shown as a black dot and the average of the standard deviation of each set of data is shown as a horizontal line.
FIG. 9 is a microscopic image of E.coli cultured with cephalexin at a concentration of 90. mu.g/mL for various periods of time; wherein a is cultured for 0 h; b is culturing for 4 hours; c is culturing for 8 h.
FIG. 10 is an electron micrograph of E.coli after cephalexin treatment; wherein the scale is 60 μm.
FIG. 11 is a statistical graph of the concentration of cefalexin required for the preparation of ultra-long E.coli from different E.coli strains.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The reagents and raw materials which are not labeled in the invention are all purchased from the market.
EXAMPLE 1 CTAB production of ultra-Long E.coli
CTAB is white or light yellow solid substance, is easily dissolved in isopropanol, is soluble in water, has good biodegradation and sterilization performance, and is prepared by water, so that the concentration of the CTAB solution in a culture medium is 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) in sequence.
② 12h culture and absorbance value of 0.5(OD 0.5) bacterial liquid using medium dilution 1000 times, diluted Escherichia coli inoculated into LB medium, 37 degrees C, 60rpm aerobic culture for 2 h.
③ inoculating the Escherichia coli re-cultured in (c) to a medium containing 0.0005% (w/w) CTAB + 90. mu.g/mL cephalexin, 0.001% (w/w) CTAB + 90. mu.g/mL cephalexin, 0.002% (w/w) CTAB + 90. mu.g/mL cephalexin, 0.005% (w/w) CTAB + 90. mu.g/mL cephalexin, 0.01% (w/w) CTAB + 90. mu.g/mL cephalexin and 0.02% (w/w) CTAB + 90. mu.g/mL cephalexin, aerobically culturing at 37 ℃ and 60rpm for 4 hours, and measuring the bacterial length after the culture. Contrast setting: negative control without cefalexin and CTAB (WT) and positive control with 90. mu.g/mL cefalexin (Cex). The experimental flow chart is shown in figure 1.
Example 2 preparation of ultra-Long Escherichia coli by SDS
SDS is a white or light yellow slightly viscous substance, is easy to dissolve in water, and has good emulsifying and decontaminating performance. Since the maximum concentration of SDS in water is limited by solubility, the concentration used is not so high, and the solutions prepared with water are made to have concentrations of 0.005% (w/w), 0.02% (w/w), 0.05% (w/w), 0.1% (w/w), 0.2% (w/w) and 0.5% (w/w), respectively, in the medium.
[ solution ] Escherichia 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 ℃ under aerobic conditions at 60rpm for 12 hours (using a 250mL Erlenmeyer flask, the same applies hereinafter).
② 12h culture and absorbance value of 0.5(OD 0.5) bacterial liquid using medium dilution 1000 times, diluted Escherichia coli inoculated into LB medium, 37 degrees C, 60rpm aerobic culture for 2 h.
③ inoculating the Escherichia coli re-cultured in (c) to a medium of 0.005% (w/w) SDS + 90. mu.g/mL cephalexin, 0.02% (w/w) SDS + 90. mu.g/mL cephalexin, 0.05% (w/w) SDS + 90. mu.g/mL cephalexin, 0.1% (w/w) SDS + 90. mu.g/mL cephalexin, 0.2% (w/w) SDS + 90. mu.g/mL cephalexin and 0.5% (w/w) SDS + 90. mu.g/mL cephalexin, aerobically culturing at 37 ℃ and 60rpm for 4 hours, and measuring the bacterial length after the culture. Contrast setting: negative control with no addition of cephalexin and SDS (WT), positive control with addition of cephalexin (Cex) at 90. mu.g/mL.
Example 3Tween-20 production of ultra-Long E.coli
Tween-20 has strong hydrophilic property due to the molecular formula containing more hydrophilic groups, is often used as an oil-in-water (O/W) emulsifier, and can be used together with other emulsifiers to enhance the stability of the emulsifiers. Tween-20 was formulated at a higher concentration than CTAB and SDS (in water) due to its low toxicity, so that it was present in the medium at six concentrations, 0.02%, 0.1%, 0.2% (w/w), 1% (w/w), 2% (w/w) and 10% (w/w), respectively.
[ solution ] Escherichia 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 ℃ under aerobic conditions at 60rpm for 12 hours (using a 250mL Erlenmeyer flask, the same applies hereinafter).
② 12h culture and absorbance value of 0.5(OD 0.5) bacterial liquid using medium dilution 1000 times, diluted Escherichia coli inoculated into LB medium, 37 degrees C, 60rpm aerobic culture for 2 h.
Thirdly, inoculating the escherichia coli which is re-cultured in the second step into 0.02% (w/w) Tween-20+90 mug/mL cephalexin, 0.1% (w/w) Tween-20+90 mug/mL cephalexin, 0.2% (w/w) Tween-20+90 mug/mL cephalexin, 1% (w/w) Tween-20+90 mug/mL cephalexin, 2% (w/w) Tween-20+90 mug/mL cephalexin and 10% (w/w) Tween-20+90 mug/mL cephalexin culture medium respectively, carrying out aerobic culture at 37 ℃ and 60rpm for 4 hours, and measuring the length of the bacteria respectively after the culture is finished. Contrast setting: negative control without cefalexin and Tween-20(WT) and positive control with 90. mu.g/mL cefalexin (Cex).
Example 4 ultra-Long E.coli characterization
(1) Calculation of average Length of ultra-Long Escherichia coli
According to the following three assumptions: 1) the surface charge of the E.coli was not changed by the surfactant, 2) the charge was uniformly distributed along the E.coli, 3) the E.coli was rod-shaped and columnar, and the average lengths of the E.coli samples (examples 1 to 3) treated with cephalexin, cephalexin and surfactant were calculated, respectively.
The calculation method comprises the following steps:
electrophoretic mobility vs e.coli charge relationship: q6 pi η r mu (1)
The zeta potential z is related to the electrophoretic mobility by:
(1) the following equations and (2) are combined to obtain the zeta potential z as a function of E.coli charge: q ═ 4 pi rzf (ka) (3)
Assuming that escherichia coli is a cylinder with diameter D, length L, and ratio n ═ L/D, the diffusion coefficient D is:
according to the einstein-stokes relationship:
combining the formulas (4) and (5) to obtain:
assuming that the charge is uniformly distributed along the E.coli, we have: q Adn (7)
The combination of the formulas (3), (6) and (7) gives:
finally obtaining the relation between the zeta potential z and the length of the escherichia coli according to the formula (8):
in the formula: 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. Is the dielectric constant of water, and f (ka) is the henry function (which is 1.5 according to the semorvue-fusi approximation). k is the boltzmann constant and T is the absolute temperature.
From the final formula, the zeta potential z is related to n, and n is L/d, so that the zeta potential z on the cell surface of E.coli is positively related to the length of the bacteria, i.e., the higher the zeta potential on the cell surface, the longer the bacteria will be
The length of E.coli prepared in example 1 is shown in FIG. 2, and it can be seen from the figure that the average length of E.coli produced with CTAB in combination with cephalexin at concentrations of 0.005% (w/w) and 0.02% (w/w) is slightly shorter than that of E.coli produced with cephalexin alone, wherein the elongation effect of 0.005% (w/w) CTAB is least desirable. 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 elongating E.coli, with the 0.001% (w/w) enhancement being the best, about 1.5 times that of cephalexin (Cex) alone.
The length of E.coli prepared in example 2 is shown in FIG. 3, and it can be seen that five different concentrations of SDS enhance the effect of cephalexin-elongating E.coli, wherein the enhancing effect of 0.5% (w/w) is least significant, and the enhancing effect of 0.1% (w/w) SDS is the best, about 2 times that of cephalexin alone. It can also be seen that the 0.05% (w/w) and 0.2% (w/w) elongation effects are about 1.8 times as much as cefalexin alone, and that the 0.005% (w/w) elongation effect is more pronounced than the 0.02% (w/w).
The length of E.coli prepared in example 3 is shown in FIG. 4, and it can be seen that different concentrations of Tween-20 have an enhancing effect on the elongation of E.coli by cephalexin, and the elongation effect is significantly different. The effect of 0.02% (w/w), 0.1% and 0.2% (w/w) Tween-20 in enhancing cefalexin and prolonging Escherichia coli is similar, and the length distribution is uniform. The 1% (w/w) and 2% (w/w) Tween-20 have significant effect on the elongation of Escherichia coli, wherein the elongation of 1% (w/w) Tween-20 is the best, which is about 3 times that of cefalexin alone.
(2) Ultra-long E.coli cell membrane integrity and DNA distribution
Surfactants have cytotoxic properties such as inducing lysis of gram positive and gram negative bacteria, inhibiting bacterial growth and altering cell wall structure and porosity. Meanwhile, cefalexin as an antibiotic has a certain bacteriostatic effect. It was therefore necessary to determine whether the bacteria treated with the surfactant in combination with cephalexin, or those treated with cephalexin, had normal cellular activity.
Firstly, collecting the Escherichia coli liquid cultured normally for 4h at 37 ℃ under the aerobic condition of 60 rpm.
② collecting Escherichia coli with 90 ug/mL cephalexin exposed for 4h (cephalexin added in LB medium) under aerobic condition of 37 ℃ and 60rpm
And thirdly, respectively collecting escherichia coli which is exposed for 4 hours (surfactant and cephalexin are added in LB culture medium) through 0.001% (W/W) CTAB +90 mug/mL cephalexin, 0.1% (W/W) SDS +90 mug/mL cephalexin and 1% (W/W) Tween-20+90 mug/mL cephalexin.
And fourthly, respectively taking 500 mu L of bacteria sample in the third step, staining the bacteria sample with 200 mu L of DAPI for 5min, centrifuging the bacteria sample at 8000rpm for 5min, and collecting the bacteria sample.
Fifthly, resuspending the bacterial particles in the fourth step in 200 mu L FM4-64 solution respectively, culturing for 1 minute at 0 ℃, centrifuging the suspension for 5 minutes at 8000rpm, collecting the bacterial particles and resuspending the bacterial particles in distilled water respectively.
Sixthly, respectively observing the bacterial cell membrane and DNA in the fifth step by using a fluorescence microscope.
As shown in FIG. 5, the DNA distribution of the ultra-long E.coli produced by the combined action of the surfactant and the antibiotic was longer than that of the normal cells and that of the ultra-long bacteria produced by the action of cephalexin, and was normally distributed in the cells and uniformly distributed in the longitudinal direction of E.coli.
The distribution of cell membranes is shown in FIG. 6, the cell membranes of the ultra-long Escherichia coli generated by the combined action of the surfactant and the antibiotic are longer than those of the normal cells and the ultra-long bacteria generated by the action of the cephalexin, and the cell membranes are continuously and uniformly distributed on the surface of the Escherichia coli, and the surface morphology is normal.
Thus, the bacteria acted on by the surfactant in combination with cephalexin had normal cellular activity.
(3) Surfactant interaction with cells
The surfactant can interact with cell membrane, promote transmembrane transport of organic matters, and improve permeability of cell membrane. Surfactants are structurally similar to lipids on cell membranes, so surfactants are likely to interact with E.coli by being heavily inserted into the cell membrane, a hypothesis that we have verified by zeta potential measurements. The zeta potential of E.coli is proportional to the charge, and when a surfactant is inserted or bound in large amounts to the cell surface (including cell membrane and cell wall), the zeta potential changes, which should be different for SDS and CTAB carrying different charges.
Firstly, collecting the Escherichia coli liquid cultured normally for 4h at 37 ℃ under the aerobic condition of 60 rpm.
② collecting Escherichia coli exposed to cefalexin at 90. mu.g/mL for 4 hours (cefalexin was added to LB medium) at 37 ℃ under aerobic condition of 60 rpm.
③ respectively collecting Escherichia coli which is exposed for 4h (surfactant and cephalexin are added in LB culture medium) by 0.001 percent (W/W) CTAB +90 mug/mL cephalexin, 0.1 percent (W/W) SDS +90 mug/mL cephalexin and 1 percent (W/W) Tween-20+90 mug/mL cephalexin under the aerobic condition of 37 ℃ and 60 rpm.
And fourthly, respectively centrifuging the escherichia coli samples in the second step and the third step for 5min at 8000rpm, collecting bacterial particles after centrifugation is finished, and re-suspending the bacterial particles in pure water.
(v) measuring the surface potential of the cell membrane of Escherichia coli in (v) with Zeta-PALS (ZETAPALS/BI-200SM, Brookhaven, USA) three times under the same experimental conditions.
As shown in FIG. 7, the Zeta potential of the cell membrane surface was lower than that of the untreated E.coli, regardless of whether the treated E.coli was treated with cephalexin alone or with a surfactant in combination with cephalexin. Meanwhile, the zeta potential of the escherichia coli treated by the cefalexin alone is obviously changed. However, the zeta potential of the surfactant and the E.coli sample treated with cephalexin almost agreed with that of the E.coli sample treated with cephalexin alone.
The above results indicate that the surfactant is not inserted or bound to the cell surface and does not alter the normal function of E.coli.
Comparative example 1 production of ultra-long E.coli by conventional technique
[ solution ] Escherichia 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 ℃ under aerobic conditions at 60rpm for 12 hours (using a 250mL Erlenmeyer flask, the same applies hereinafter).
② 12h culture and absorbance value of 0.5(OD 0.5) bacterial liquid using medium dilution 1000 times, diluted Escherichia coli inoculated into LB medium, 37 degrees C, 60rpm aerobic culture for 2 h.
③ inoculating the bacteria in the second step into LB culture medium of 80 mug/mL cephalexin, 90 mug/mL cephalexin and 100 mug/mL cephalexin respectively, aerobically culturing for 4h at 37 ℃ and 60rpm, and measuring the length of the bacteria after the culture is finished.
Fourthly, respectively inoculating the bacteria in the second step into 80 mu g/mL cephalexin, 90 mu g/mL cephalexin and 100 mu g/mL cephalexin LB culture media, carrying out aerobic culture at 37 ℃ and 60rpm for 8h, and measuring the length of the bacteria after the culture is finished.
Fifthly, respectively randomly extracting 50 strains of escherichia coli treated by cephalexin with different concentrations from each group, and measuring the length by adopting an electron microscope. The length calculation method was the same as in example 4, step (1).
As a result, as shown in FIGS. 8 and 9, the optimum concentration of cephalexin was 90. mu.g/mL, and the optimum treatment time was 4 hours. The number of cells after culture is reduced because cefalexin has certain cytotoxicity. Too long incubation time (8 hours), depletion of cefalexin, and a decrease in concentration resulted in disappearance of prolonged results (fig. 9 c). The concentration of the cefalexin is the key for obtaining the ultra-long escherichia coli, and the control needs to be realized through accurate culture time control, namely, the escherichia coli is gradually increased along with the extension of the culture time, and meanwhile, the amount of the cefalexin in the escherichia coli is gradually reduced. During the first four hours, the amount of cephalexin is still sub-lethal, as evidenced by the fact that E.coli is still growing. After four hours, the E.coli cells were gradually recovered as the amount of cefalexin decreased beyond the effective concentration. The attenuation time of cefalexin is strictly controlled, so that the ultra-long escherichia coli can be obtained to the maximum extent. In addition, electron microscope measurement is carried out on the ultra-long escherichia coli (obtained in the third step) prepared by the traditional method, and the measurement result shows that the ultra-long escherichia coli is in a long filament shape with the width of about 1 micron, and the length of the ultra-long escherichia coli is nearly hundred times longer than that of common escherichia coli (figure 10). It was also found that the very long E.coli only occupies a small fraction of the total amount of all E.coli, and that the vast majority of E.coli is still in an unextended state.
Comparative example 2
The speed of bacteria shaking in the third step of the embodiment 1 is changed into 0rpm, 30rpm and 200rpm respectively, and other conditions are completely the same, so that the ultra-long escherichia coli is prepared. The container for shaking the bacteria in the third step of the example 1 is changed into a 50mL centrifuge tube, a 500mL conical flask and a 1000mL conical flask respectively, and the rest conditions are completely the same, so that the ultra-long escherichia coli is prepared.
As a result, it was confirmed that the growth of Escherichia coli was slow due to insufficient number of revolutions during shaking or static culture (number of revolutions: 0) and that a sufficient amount of Escherichia coli could not be obtained; on the other hand, too high a bacterial shaking speed results in a shorter longest length of E.coli. Along with the increase of the caliber of the bacteria container, the mechanical shearing force effect generated by the same revolution is greatly different. At about 60rpm, the ultra-long Escherichia coli can be prepared by shaking the bacteria in a 250mL conical flask. If a bacterium container with a larger caliber is used, the rotation number is reduced so as to prevent the death of bacteria or the breakage of ultra-long escherichia coli caused by excessive mechanical collision; if a fungus container with a smaller caliber is used, the rotation number should be increased correspondingly.
Comparative example 3
Escherichia coli (E.coli pD1B10, E.coli BL21, E.coli MG1655 strain) was inoculated into LB medium containing 10g/L sodium chloride, 5g/L yeast extract and 10g/L tryptone, respectively, at pH 7.0, and cultured overnight at 37 ℃ and 60rpm in the presence of oxygen for 12 hours (using a 250mL Erlenmeyer flask, the same applies below).
② 12h culture and absorbance value of 0.5(OD 0.5) bacterial liquid using medium dilution 1000 times, diluted Escherichia coli inoculated into LB medium, 37 degrees C, 60rpm aerobic culture for 2 h.
③ inoculating the bacterium of step II to LB culture medium of cephalexin of 30 ug/mL, 45 ug/mL, 60 ug/mL, 75 ug/mL, 90 ug/mL, 105 ug/mL and 120 ug/mL, aerobically culturing at 37 ℃ and 60rpm for 4h, and measuring the length of the bacterium after the culture.
The results are shown in FIG. 11, where different E.coli strains have different concentrations of cefalexin to be used for preparing the over-length E.coli.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the 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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Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54126722A (en) * | 1978-03-23 | 1979-10-02 | Toyo Jozo Co Ltd | Slow release antibiotic tablet |
| GB1598062A (en) * | 1977-03-31 | 1981-09-16 | Sanraku Ocean Co | Antibiotic and its antibacterial derivatives |
| US20010013931A1 (en) * | 2000-02-14 | 2001-08-16 | Shimadzu Corporation | Double beam spectrophotometer |
| CN1390134A (en) * | 1999-09-14 | 2003-01-08 | 新眼界诊断公司 | The use of bacterial phage associated lysing enzymes for the prophylactic and therapeutic treatment of various illnesses |
| US20030096766A1 (en) * | 1996-01-25 | 2003-05-22 | Mclean Allan Joseph | Methods of and compositions for potentiating the action of agents active on cell wall sites of the susceptible bacteria |
| EP1750688A1 (en) * | 2004-04-30 | 2007-02-14 | Allergan, Inc. | Steroid intraocular implants having an extended sustained release for a period of greater than 2 months |
| US8163896B1 (en) * | 2002-11-14 | 2012-04-24 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory genes and uses thereof |
| WO2014179784A2 (en) * | 2013-05-03 | 2014-11-06 | Microbiotix, Inc. | Antimicrobial potentiators |
| EP2970921A1 (en) * | 2013-03-15 | 2016-01-20 | aTyr Pharma, Inc. | Histidyl-trna synthetase-fc conjugates |
| CN105820053A (en) * | 2016-04-23 | 2016-08-03 | 黄亦琼 | Pharmaceutical composition of cefalexin and application of pharmaceutical composition in biological medicine |
| TW201741331A (en) * | 2016-03-30 | 2017-12-01 | 康華特科技有限公司 | Detecting microbial infection in wounds |
| TW201800753A (en) * | 2016-03-30 | 2018-01-01 | 奎立茲梅診斷有限兩合公司 | Detecting microbial infection in wounds |
| CN107656056A (en) * | 2017-08-29 | 2018-02-02 | 山东师范大学 | A kind of method increased based on bacterium to the quick microscopy of bacterium |
| EP3403097A1 (en) * | 2016-01-15 | 2018-11-21 | DSM IP Assets B.V. | Method for detecting an analyte |
| US20190085034A1 (en) * | 2016-03-16 | 2019-03-21 | Tarveda Therapeutics, Inc. | Antibody mimic conjugates and particles |
| WO2019142002A1 (en) * | 2018-01-19 | 2019-07-25 | Ucl Business Plc | Compositions for the treatment of a disease of the urinary tract and treatment of a disease involving the intracellular delivery of the particle or a medicament contained therein |
| CN112326607A (en) * | 2020-10-16 | 2021-02-05 | 暨南大学 | Low-concentration ROS detection method and application thereof |
-
2020
- 2020-07-24 CN CN202010721564.XA patent/CN111826334A/en active Pending
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1598062A (en) * | 1977-03-31 | 1981-09-16 | Sanraku Ocean Co | Antibiotic and its antibacterial derivatives |
| JPS54126722A (en) * | 1978-03-23 | 1979-10-02 | Toyo Jozo Co Ltd | Slow release antibiotic tablet |
| US20030096766A1 (en) * | 1996-01-25 | 2003-05-22 | Mclean Allan Joseph | Methods of and compositions for potentiating the action of agents active on cell wall sites of the susceptible bacteria |
| CN1390134A (en) * | 1999-09-14 | 2003-01-08 | 新眼界诊断公司 | The use of bacterial phage associated lysing enzymes for the prophylactic and therapeutic treatment of various illnesses |
| US20010013931A1 (en) * | 2000-02-14 | 2001-08-16 | Shimadzu Corporation | Double beam spectrophotometer |
| US8163896B1 (en) * | 2002-11-14 | 2012-04-24 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory genes and uses thereof |
| EP1750688A1 (en) * | 2004-04-30 | 2007-02-14 | Allergan, Inc. | Steroid intraocular implants having an extended sustained release for a period of greater than 2 months |
| EP2970921A1 (en) * | 2013-03-15 | 2016-01-20 | aTyr Pharma, Inc. | Histidyl-trna synthetase-fc conjugates |
| WO2014179784A2 (en) * | 2013-05-03 | 2014-11-06 | Microbiotix, Inc. | Antimicrobial potentiators |
| EP3403097A1 (en) * | 2016-01-15 | 2018-11-21 | DSM IP Assets B.V. | Method for detecting an analyte |
| US20190085034A1 (en) * | 2016-03-16 | 2019-03-21 | Tarveda Therapeutics, Inc. | Antibody mimic conjugates and particles |
| TW201741331A (en) * | 2016-03-30 | 2017-12-01 | 康華特科技有限公司 | Detecting microbial infection in wounds |
| TW201800753A (en) * | 2016-03-30 | 2018-01-01 | 奎立茲梅診斷有限兩合公司 | Detecting microbial infection in wounds |
| CN105820053A (en) * | 2016-04-23 | 2016-08-03 | 黄亦琼 | Pharmaceutical composition of cefalexin and application of pharmaceutical composition in biological medicine |
| CN107656056A (en) * | 2017-08-29 | 2018-02-02 | 山东师范大学 | A kind of method increased based on bacterium to the quick microscopy of bacterium |
| WO2019142002A1 (en) * | 2018-01-19 | 2019-07-25 | Ucl Business Plc | Compositions for the treatment of a disease of the urinary tract and treatment of a disease involving the intracellular delivery of the particle or a medicament contained therein |
| CN112326607A (en) * | 2020-10-16 | 2021-02-05 | 暨南大学 | Low-concentration ROS detection method and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| MICHAEL T. DAVISON 等: "Immunochemical Demonstration of Zonal Growth of the Cell Envelope of Escherichia coli", 《EUR.J.BIOCHEM.》 * |
| SEN HOU等: "Joint effect of surfactants and cephalexin on the formation of Escherichia coli filament", 《ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY》 * |
| TAKESHI NISHINO 等: "Morphological Changes in Staphylococcus aureus and Escherichia coli Exposed to Cephalexin", 《JAPAN.J.MICRIBIOL》 * |
| 何东平 主编: "《油脂工厂设计》", 30 April 2017, 中国轻工业出版社 * |
| 张志林 等: "β内酞胺类抗生素引起的大肠杆菌形态改变", 《上海第一医学院学报》 * |
| 王丽平 等: "不同抗生素对大肠杆菌形态的影响及其诱导游离内毒素的释放", 《中国兽医学报》 * |
| 陈大明等: "合成生物学应用产品开发现状与趋势", 《中国生物工程杂志》 * |
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