Method for modifying bacteria through oil-free mild aqueous two-phase strategy

Abstract

The invention discloses a method for modifying bacteria through an oil-free mild aqueous two-phase strategy. The method has relatively high biocompatibility; no mechanical force is introduced, and no damage is caused to bacteria; the method is suitable for modification of various bacteria and has universality. The method disclosed by the invention is simple, convenient and rapid to operate, low in cost and more beneficial to popularization, and has important significance on drug delivery based on bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for modifying bacteria using an oil-free and mild two-aqueous phase strategy. Background Art

[0002] Microorganisms are the largest species resource and gene resource library on the earth. The metabolite library formed by the physiologically active substances and their derivatives produced by microorganisms in their life activities plays a huge role in the field of medical health. Humans have used microorganisms and their metabolites to treat diseases for thousands of years. Human health is mainly affected by the human genome, living environment and human microorganisms. Compared with the genome and living environment that cannot be selected, microorganisms have become a more critical and controllable factor for us to change our own health status. With the in-depth study of human microbiota, more and more intervention measures have been verified and are expected to be applied in clinical treatment. At present, the use of bacteria and their biological components as a therapeutic means shows great potential in the treatment of diseases. Compared with injectable bacteria, oral administration of bacteria can improve patient compliance and is considered the preferred mode.

[0003] However, due to the harsh gastrointestinal environment, the survival ability and therapeutic effect of the target microorganisms in the body are not ideal after direct oral administration. In recent years, with the rapid development of synthetic biology and nanotechnology, bacteria and biological components have been designed to achieve targeted gene reprogramming and precise spatiotemporal control in the gastrointestinal tract to improve their survival ability and therapeutic efficiency. At present, the delivery of engineered bacteria is mainly divided into surface modification of bacteria and genetic engineering. For bacterial surface modification, a variety of materials have been developed, but the modification methods are still mostly concentrated on traditional methods such as soaking and stirring. However, the soaking and stirring methods cannot better control the uniformity, the number of encapsulated bacteria, and the thickness of the surface modification layer, and they require the introduction of mechanical force, which is not gentle enough.

[0004] At present, new coating methods have emerged, such as surface modification of bacteria through microfluidic devices. However, microfluidic encapsulation systems are expensive, pipes are easily clogged, and face problems such as low encapsulation efficiency. In addition, microfluidic strategies often cannot avoid the introduction of substances with low biocompatibility such as oils and surfactants, and the degreasing step will significantly reduce the activity of bacteria and prolong the modification time and increase the complexity of the operation, further limiting its wide application in the biomedical field. Therefore, developing a controllable and uniform bacterial surface modification method is a scientific problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide an oil-free and mild method for modifying bacteria using a two-aqueous phase strategy.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for modifying bacteria using an oil-free and mild two-aqueous phase strategy, comprising:

[0009] Bacteria were cultured overnight, centrifuged, the supernatant was discarded, an equal volume of PBS buffer was added to resuspend, and centrifuged three times. The precipitate after the last centrifugation was resuspended in PBS buffer to obtain bacterial solution;

[0010] Mix the bacterial solution with the upper solution to obtain an upper bacterial suspension;

[0011] Slowly add the lower solution to the container, then slowly add the upper bacterial suspension to form a two-phase aqueous system with stable upper and lower stratifications. Let it stand for a while until the bacteria fall naturally.

[0012] After the upper layer solution is allowed to stand for solidification, the upper layer is taken out, the lower layer is allowed to solidify, and the precipitate is collected after centrifugation. The precipitate is the bacteria modified by the oil-free and gentle two-phase aqueous strategy.

[0013] As a preferred embodiment of the method of the present invention, the upper layer solution is one of a sodium alginate solution, a carboxymethyl cellulose solution, a matrigel solution, a methacrylic anhydride gelatin solution, and a sericin solution.

[0014] As a preferred embodiment of the method of the present invention, the concentration of the sodium alginate solution, carboxymethyl cellulose solution and matrigel solution is 0.001-1%; the concentration of the methacrylic anhydride gelatin solution and sericin solution is 10%.

[0015] As a preferred embodiment of the method of the present invention, the lower layer solution is one of a dextran solution, a polysucrose solution, a sucrose solution and an agarose solution.

[0016] As a preferred solution of the method of the present invention, the concentration of the lower layer solution is 10%.

[0017] As a preferred embodiment of the method of the present invention, the curing is to add a curing agent or perform a corresponding curing operation.

[0018] As a preferred embodiment of the method of the present invention, the solidification reagent is a calcium chloride solution or a ferric chloride solution.

[0019] As a preferred embodiment of the method of the present invention, the solidification operation is to absorb and discard the upper layer solution and then irradiate with light of corresponding wavelength.

[0020] As a preferred embodiment of the method of the present invention, the centrifugal speed is 5500 rpm.

[0021] As a preferred embodiment of the method of the present invention, the bacteria is one of Escherichia coli, yeast, Gram-positive bacteria and Gram-negative bacteria.

[0022] Beneficial effects of the present invention:

[0023] (1) The method of the present invention has good uniformity and can produce a large number of single bacterial individuals with complete surface coating;

[0024] (2) The modification method of the present invention does not introduce chemical substances harmful to bacterial growth or organic phases with poor biocompatibility, does not occupy sites on the surface of bacterial cell membranes, and has a mild action force with almost no damage to the bacteria;

[0025] (3) The present invention is applicable to a variety of bacteria, including Gram-positive and Gram-negative bacteria;

[0026] (4) The external material used in the present invention is optional, and other substances (growth factors, signal factors, etc.) can be added to the external material.

[0027] (5) The aqueous two-phase modification strategy proposed in the present invention has the advantages of mild action, high biocompatibility, no restriction on bacterial species, simple operation, and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0029] Figure 1 Schematic diagram of bacterial surface modification based on a mild, oil-free aqueous two-phase strategy according to the present invention.

[0030] Figure 2 The stratification of different solutions.

[0031] Figure 3 Preparation of surface modified Escherichia coli Nissle 1917 for different solution combinations.

[0032] Figure 4 To modify the surfaces of different types of bacteria using a mild, oil-free aqueous two-phase strategy.

[0033] Figure 5 Plate coating results before and after bacterial surface modification using a mild, oil-free aqueous two-phase strategy. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] Materials and reagents used in the examples of the present invention: Alginate (AR grade) (Sigma), CMC V (Aladdin, JAD-C104983), Matrigel (corning, 356234), GelMA, Sericin, calcium chloride, Dextran (Aladdin, D490149-750K), Ficoll (Sigma, F2637), electron microscope copper mesh, 0.22 μm filter membrane, and magnetic rotor.

[0038] Example 1

[0039] (1) Prepare 0.01% Alginate solution and 10% Dextran solution

[0040] Accurately weigh 1 mg of sodium alginate solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve;

[0041] Accurately weigh 1 g of dextran solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0042] (2) Preparation of calcium chloride and ferric chloride solution

[0043] Prepare a 2% calcium chloride solution by accurately weighing 2g of calcium chloride and dissolving it in 100ml of distilled water to obtain a 2% calcium chloride solution.

[0044] Prepare a 2% ferric chloride solution by accurately weighing 2 g of ferric chloride and dissolving it in 100 ml of distilled water to obtain a 1% ferric chloride solution.

[0045] (3) Escherichia coli Nissle1917 (purchased from ATCC) was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0046] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and then use 1ml of the target CFU=10 10 The target strain was thoroughly mixed with 500 μl of 0.01% Alginate solution, and bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0047] Slowly add 10% Dextran solution to the lower layer of the cuvette, aspirate the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 6 hours, aspirate 400 μl of the upper layer solution with a needle, and then add 2% CaCl 2 After the remaining Alginate solidifies, pick out the solidified Alginate with a needle, draw the lower layer of solution into a new 2 ml EP tube, and add an equal volume of 2% CaCl 2 The solution was mixed and centrifuged, and the precipitate was the target strain modified with Alginate.

[0048] Schematic diagram of the process of bacterial surface modification based on the mild and oil-free aqueous two-phase strategy. Figure 1 .

[0049] Example 2

[0050] (1) Prepare 0.01% CMC V solution and 10% Dextran solution

[0051] Accurately weigh 1 mg of carboxymethyl cellulose (CMC V) solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve;

[0052] Accurately weigh 1 g of dextran solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0053] (2) Preparation of calcium chloride and ferric chloride solution

[0054] Prepare a 2% calcium chloride solution by accurately weighing 2g of calcium chloride and dissolving it in 100ml of distilled water to obtain a 2% calcium chloride solution.

[0055] Prepare a 2% ferric chloride solution by accurately weighing 2 g of ferric chloride and dissolving it in 100 ml of distilled water to obtain a 1% ferric chloride solution.

[0056] (3) Escherichia coli Nissle1917 was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0057] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and then use 1ml of the target CFU=10 10 The target strain was thoroughly mixed with 500 μl of 0.01% CMC V solution, and the bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0058] Slowly add 10% Dextran solution to the lower layer of the cuvette, aspirate the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 6 hours, aspirate 400 μl of the upper layer solution with a needle, and then add 2% CaCl 2 After the remaining CMC V was solidified, the solidified CMC V was picked out with a needle, and the lower layer of solution was drawn into a new 2 ml EP tube, and an equal volume of 2% CaCl 2 The solution was mixed and centrifuged, and the precipitate was the target strain after CMC V modification.

[0059] Example 3

[0060] (1) Prepare 0.01% Matrigel solution and 10% Dextran solution

[0061] Accurately weigh 1 μl of Matrigel solution, dissolve it in 10 ml of filtered MQ, add the rotor, and place it on a magnetic stirrer to dissolve;

[0062] Accurately weigh 1 g of dextran solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0063] (2) Preparation of calcium chloride and ferric chloride solution

[0064] Prepare a 2% calcium chloride solution by accurately weighing 2g of calcium chloride and dissolving it in 100ml of distilled water to obtain a 2% calcium chloride solution.

[0065] Prepare a 2% ferric chloride solution by accurately weighing 2 g of ferric chloride and dissolving it in 100 ml of distilled water to obtain a 1% ferric chloride solution.

[0066] (3) Escherichia coli Nissle1917 was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0067] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and use the target 1ml CFU=10 10 The target strain was mixed with 500 μl of 0.01% Matrigel solution, and the bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0068] Slowly add 10% Dextran solution to the lower layer of the cuvette, aspirate the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 6 hours, aspirate 400 μl of the upper layer solution with a needle, and then add 2% CaCl 2 After the remaining Matrigel solidifies, pick out the solidified Matrigel with a needle, draw the lower layer of solution into a new 2 ml EP tube, and add an equal volume of 2% CaCl 2 The solution was mixed and centrifuged, and the precipitate was the target strain modified with Matrigel.

[0069] Example 4

[0070] The entire experimental process of this embodiment needs to be operated in a dark environment.

[0071] (1) Preparation of 10% GelMA solution

[0072] Weigh the corresponding amount of GelMA, place it on a magnetic stirrer in a dark place to dissolve and prepare the GelMA solution, add 1% inulin to the solution and stir well;

[0073] A photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), was added to the above solution to prepare a GelMA solution containing LAP.

[0074] (2) Prepare 10% Dextran solution

[0075] Accurately weigh 1 g of dextran solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0076] (3) Escherichia coli Nissle1917 was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0077] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and use the target 1ml CFU=10 10 The target strain was thoroughly mixed with 500 μl of 10% GelMA solution, and the bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0078] Slowly add 10% Dextran solution to the lower layer of the cuvette, absorb the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 3 hours, use a long needle to penetrate the upper solution and absorb the lower layer, be careful not to absorb the upper solution. Transfer the absorbed lower solution to a 2ml clean EP tube and irradiate it with ultraviolet light for 5 minutes. Centrifuge at 12000rpm for 5 minutes, and the precipitate is the target strain modified with GelMA.

[0079] Example 5

[0080] The entire experimental process of this embodiment needs to be operated in a dark environment.

[0081] (1) Preparation of 10% Sericin solution

[0082] 1 g of sericin solid was accurately weighed, dissolved in 10 ml of filtered MQ, added with a rotor, and placed on a magnetic stirrer for dissolution.

[0083] (2) Prepare 10% Dextran solution

[0084] Accurately weigh 1 g of dextran solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0085] (3) Preparation of calcium chloride and ferric chloride solution

[0086] Prepare a 1% calcium chloride solution by accurately weighing 1g of calcium chloride and dissolving it in 100ml of distilled water to obtain a 1% calcium chloride solution.

[0087] Prepare a 1% ferric chloride solution by accurately weighing 1g of ferric chloride and dissolving it in 100ml of distilled water to obtain a 1% ferric chloride solution.

[0088] (4) Escherichia coli Nissle1917 was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0089] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and then use 1ml of the target CFU=10 10 The target strain was thoroughly mixed with 500 μl of 10% Sericin solution, and the bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0090] Slowly add 10% Dextran solution to the lower layer of the cuvette, absorb the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 3 hours, use a long needle to penetrate the upper solution and absorb the lower layer, be careful not to absorb the upper solution. Transfer the absorbed lower solution to a 2ml clean EP tube, irradiate with green light for 5 minutes. Centrifuge at 12000rpm for 5 minutes, and the precipitate is the target strain modified by Sericin.

[0091] The stratification of 0.01% Alginate solution, CMC V solution, Matrigel solution and 10% Dextran solution prepared in Examples 1 to 5, and the stratification of 10% GelMA solution and Sericin solution and 10% Dextran solution, respectively, are shown in FIG. Figure 2 As shown, it can be seen that all of them are stably stratified; among them, (a) is the stratification of Alginate solution and Dextran solution, (b) is the stratification of CMC V solution and Dextran solution, (c) is the stratification of Matrigel solution and Dextran solution, (d) is the stratification of GelMA solution and Dextran solution, and (e) is the stratification of Sericin solution and Dextran solution.

[0092] In Examples 1 to 5, Alginate solution, CMC V solution, Matrigel solution, GelMA solution, Sericin solution and Dextran solution were respectively combined to prepare surface-modified Escherichia coli Nissle1917. Figure 3 As shown, it can be seen that different combinations have good modification effects on Escherichia coli Nissle1917.

[0093] Example 6

[0094] The entire experimental process of this embodiment needs to be operated in a dark environment.

[0095] (1) Preparation of GelMA solution

[0096] Weigh the corresponding amount of GelMA, place it on a magnetic stirrer in a dark place to dissolve and prepare the GelMA solution, add 1% inulin to the solution and stir well;

[0097] A photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), was added to the above solution to prepare a GelMA solution containing LAP.

[0098] (2) Prepare 10% Sucrose solution

[0099] Accurately weigh 1 g of Sucrose solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0100] (3) Yeast (purchased from ATCC) was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0101] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and then use 1ml of the target CFU=10 10 The target strain was thoroughly mixed with 500 μl of 10% GelMA solution, and the bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0102] Slowly add 10% Sucrose solution to the lower layer of the cuvette, absorb the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 3 hours, use a long needle to penetrate the upper solution and absorb the lower layer, be careful not to absorb the upper solution. Transfer the absorbed lower solution to a 2ml clean EP tube and irradiate it with ultraviolet light for 5 minutes. Centrifuge at 12000rpm for 5 minutes, and the precipitate is the target strain modified with GelMA.

[0103] Example 7

[0104] The entire experimental process of this embodiment needs to be operated in a dark environment.

[0105] (1) Preparation of 10% Sericin solution

[0106] Accurately weigh 1 g of Sericin solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0107] (2) Prepare 10% Ficoll solution

[0108] Accurately weigh 1 g of Ficoll solid, dissolve it in 10 ml of filtered MQ, add a rotor, and place it on a magnetic stirrer to dissolve.

[0109] (3) Preparation of calcium chloride and ferric chloride solution

[0110] Prepare a 1% calcium chloride solution by accurately weighing 1g of calcium chloride and dissolving it in 100ml of distilled water to obtain a 1% calcium chloride solution.

[0111] Prepare a 1% ferric chloride solution by accurately weighing 1g of ferric chloride and dissolving it in 100ml of distilled water to obtain a 1% ferric chloride solution.

[0112] (4) Escherichia coli Nissle1917 was cultured overnight and centrifuged at 5500 rpm for 5 min at room temperature; the supernatant was discarded, and an equal volume of PBS buffer was added to resuspend the culture, and centrifuged at 5500 rpm for 5 min at room temperature. This step was repeated three times, and the precipitate after the last centrifugation was resuspended in PBS buffer;

[0113] UV spectrophotometer adjusts OD 600 =1.0, collect the bacterial solution and centrifuge it, and then use 1ml of the target CFU=10 10 The target strain was thoroughly mixed with 500 μl of 10% Sericin solution, and the bubbles were expelled after ultrasonication to obtain the upper bacterial suspension;

[0114] Slowly add 10% Ficoll solution to the lower layer of the cuvette, absorb the mixed bacterial suspension, slowly add it to the upper layer of the same cuvette, let it stand for 3 hours, use a long needle to penetrate the upper solution and absorb the lower layer, be careful not to absorb the upper solution. Transfer the absorbed lower solution to a 2ml clean EP tube, irradiate with green light for 5 minutes. Centrifuge at 12000rpm for 5 minutes, and the precipitate is the target strain modified by Sericin.

[0115] Example 8

[0116] The difference between this embodiment and embodiment 1 is that the encapsulated bacteria in step (3) are Gram-positive bacteria Lactobacilus rhamnosus and Gram-negative bacteria Escherichiacoli Nissle 1917, respectively, and the remaining steps are the same as those in embodiment 1.

[0117] Figure 4 In order to utilize the mild and oil-free two-phase aqueous strategy to modify the surfaces of different types of bacteria, the systems used in the figure are Alginate and Dextran as the upper phase and lower phase, respectively. Gram-positive bacteria are represented by Lactobacilus rhamnosus (left), and Gram-negative bacteria are represented by EscherichiacoliNissle 1917 (right).

[0118] Figure 5 The unmodified bacterial solution and the modified bacterial solution were diluted to the same multiple before and after the bacterial surface modification using a mild oil-free two-phase strategy, and then plated in an ultra-clean workbench. The left side (a) is the unmodified EscherichiacoliNissle 1917 (control group), and the right side (b) is the EscherichiacoliNissle 1917 after surface modification with Alginate (experimental group). (c) is the number of viable bacteria in the control group and the experimental group. The results show that the bacterial surface modification based on this method does not affect bacterial growth. It can be seen that this surface modification method has no effect on the activity of bacteria.

[0119] Comparative Example 1

[0120] The difference between this comparative example and Example 1 is that: 10% Alginate solution was prepared, and the other steps were the same as those in Example 1; and it was found that the bacteria could not be successfully modified.

[0121] Comparative Example 2

[0122] The difference between this comparative example and Example 1 is that: a 1% Dextran solution is prepared, and the other steps are the same as those of Example 1; it is found that the stratification is unstable and the upper and lower layer solutions are miscible.

[0123] Comparative Example 3

[0124] The difference between this comparative example and Example 4 is that: a 1% GelMA solution was prepared, and the other steps were the same as those in Example 1; it was found that the bacteria fell instantly and were not completely modified.

[0125] The method of the present invention has good uniformity and can produce a large number of single bacterial individuals with complete surface coating; through this modification method, no chemical substances harmful to bacterial growth or organic phases with poor biocompatibility are introduced, the sites on the surface of bacterial cell membranes are not occupied, and the force is mild and has almost no damage to bacteria; and the present invention is applicable to a variety of bacteria, including Gram-positive and Gram-negative bacteria, etc. The external materials used are selective, and other substances (growth factors, signal factors, etc.) can be added to the external materials. The two-phase aqueous modification strategy proposed by the present invention has the advantages of mild action, high biocompatibility, no restriction on bacterial species, simple operation, and large-scale production.

[0126] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. An oil-free and mild two-phase aqueous strategy for modifying bacteria. Features: include, Bacteria were cultured overnight, centrifuged, the supernatant was discarded, an equal volume of PBS buffer was added to resuspend, and centrifuged three times. The precipitate after the last centrifugation was resuspended in PBS buffer to obtain bacterial solution; Mix the bacterial solution with the upper solution to obtain an upper bacterial suspension; Slowly add the lower solution to the container, then slowly add the upper bacterial suspension to form a two-phase aqueous system with stable upper and lower stratifications. Let it stand for a while until the bacteria fall naturally. After the upper layer solution is allowed to stand for solidification, the upper layer is taken out, the lower layer is allowed to solidify, and the precipitate is collected after centrifugation. The precipitate is the bacteria modified by the oil-free and gentle two-phase aqueous strategy.

2. The method according to claim 1, Features: The upper layer solution is one of a sodium alginate solution, a carboxymethyl cellulose solution, a matrigel solution, a methacrylic anhydride gelatin solution, and a sericin solution.

3. The method according to claim 2, Features: The concentrations of the sodium alginate solution, carboxymethyl cellulose solution and matrix glue solution are 0.001-1%; the concentrations of the methacrylic anhydride gelatin solution and sericin solution are 10%.

4. The method according to claim 1, Features: The lower layer solution is one of a dextran solution, a polysucrose solution, a sucrose solution and an agarose solution.

5. The method according to claim 1, Features: The concentration of the lower layer solution is 10%.

6. The method according to claim 1, Features: The curing is to add a curing agent or perform a corresponding curing operation.

7. The method according to claim 6, Features: The curing agent is a calcium chloride solution or a ferric chloride solution.

8. The method according to claim 1, Features: The solidification operation is to absorb and discard the upper solution and then irradiate with light of corresponding wavelength.

9. The method according to claim 1, Features: The centrifugal speed was 5500 rpm.

10. The method according to claim 1, Features: The bacterium is one of Escherichia coli, yeast, Gram-positive bacteria and Gram-negative bacteria.

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