Single bacterium screening and culturing method based on digital microfluidic system

Abstract

The invention relates to a single bacterium screening and culturing method based on a digital micro-fluidic system, which comprises the following steps: separating on a chip of the digital micro-fluidic system to obtain single bacterium liquid drops; the method comprises the following steps: injecting a screened substance on a chip of a digital microfluidic system, separating to obtain single-electrode screened substance liquid drops, controlling the single-electrode screened substance liquid drops, mixing with single bacterium liquid drops to obtain mixed liquid drops, setting temperature and time, culturing bacteria in the mixed liquid drops, injecting new bacterium liquid, and repeatedly culturing. According to the present invention, after the single bacterium liquid drops are efficiently obtained with high throughput, the bacteria are screened, and the separated single bacterium liquid drops can be subjected to the in-situ repeated culture on the chip so as to achieve the proliferation of the bacteria without transferring to the outside of the chip, such that the obtained single bacteria cannot grow due to other transfer reasons, and the obtained single bacteria can be easily obtained. Finally, the sample is lost, and the pollution rate is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a single bacteria screening and culturing method based on a digital microfluidic system. Background Art

[0002] In the past 10 years, the application of microfluidics in the field of biotechnology has become relatively mature. For example, Bio-Rad relies on a microfluidics platform to develop a digital PCR chip that can achieve absolute quantitative detection of nucleic acids; the American single-cell sequencing company 10XGenomics uses a similar oil-in-water method to generate single cell samples for library construction and sequencing.

[0003] Microfluidic systems can integrate multiple sorting methods based on the physical parameters of cells. The micro size of the microfluidic system provides a perfect interface for the operation of single cells and can be adjusted and controlled in a variety of ways. Microfluidic systems can also reduce the amount of experimental reagents and reduce experimental costs, and have potential portability. Moreover, unlike traditional microfluidic systems, the encapsulation of bacteria in aqueous droplets is achieved through the oil-in-water method. Digital microfluidic systems can directly manipulate droplets containing bacteria and separate a large number of small droplets, thereby achieving high-throughput separation of single bacteria. Based on these advantages, microfluidic technology has become the best candidate for high-throughput cell sorting technology in the future. However, at this stage of digital microfluidic technology, there are still various technologies that need to be improved. For example, the existing digital microfluidic technology has a low success rate for single bacterial separation and low throughput, and it is impossible to complete high-throughput effective single bacterial separation at one time, and it is impossible to recover and re-separate bacteria that have failed single separation. With the improvement of technology, even if the problem of separation flux has been solved to a certain extent, due to the characteristics of the system, the bacteria that failed to be separated cannot be recovered and re-separated, so the sample loss rate is relatively high. The existing microfluidic system is basically unable to screen and continuously culture the single bacteria separated on the chip in situ, that is, it is impossible to perform in situ screening and continuous culture, and can only be transferred outside the chip for culture and identification, resulting in the inability to perform subsequent complex reaction operations, the screening conditions that can be used are single, and the contamination rate is relatively high. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a single bacteria screening and cultivation method based on a digital microfluidic system, which can quickly screen the required bacteria, and better cultivate anaerobic or aerobic bacteria on the same platform, and can realize in situ continuous cultivation of bacteria.

[0005] The technical solutions for achieving the above-mentioned purpose include the following.

[0006] The first aspect of the present invention is to provide a single bacteria screening and culturing method based on a digital microfluidic system, the single bacteria screening and culturing method is performed on a digital microfluidic chip, the digital microfluidic chip comprises an upper electrode plate and a lower electrode plate, a fluid channel layer is formed between the upper electrode plate and the lower electrode plate; the lower electrode plate comprises an electrode layer, the electrode layer comprises a plurality of electrodes arranged in an array;

[0007] The single bacteria screening and culturing method comprises the following steps:

[0008] S1. Inject the diluted bacterial solution into the digital microfluidic chip, control the power on and off of the electrodes on the digital microfluidic chip to form single-electrode droplets, detect and judge the single-electrode droplets, mark the droplets containing only one bacterium, and obtain single-bacteria droplets;

[0009] S2. Injecting the screening material onto the chip of the digital microfluidic system, separating and obtaining single-electrode screening material droplets, controlling the single-electrode screening material droplets, and mixing them with single bacterial droplets to obtain mixed droplets -;

[0010] S3. Setting the culture temperature, culturing the bacteria in the mixed droplets, and screening the bacteria;

[0011] S4. The culture medium is then injected into the digital microfluidic chip, and by controlling the power on and off of the electrodes on the digital microfluidic chip, a single-electrode culture medium droplet is formed, which is mixed with the bacterial droplet to continue the culture.

[0012] According to actual needs, the following steps may be optionally included:

[0013] S5. Continue culturing for a period of time. After the bacteria have multiplied for at least one generation, add a droplet of new culture medium, and tear the bacterial droplet into half of equal volume by controlling the power on and off of the electrodes on the digital microfluidic chip to obtain a bacterial droplet injected with the new culture medium;

[0014] S6. Repeat step S5 0 or more times to achieve continuous perfusion culture of bacteria on the microfluidic chip.

[0015] According to the experimental requirements, subsequent experiments can be performed on the chip or output from the chip at different time points on the torn bacterial droplets (including single bacterial droplets).

[0016] In some embodiments, the bacteria are oxygen-tolerant or anaerobic bacteria, and nitrogen is continuously passed through the chip culture chamber before screening and continuous culture to adjust the culture environment to an anaerobic state.

[0017] In some embodiments, the bacteria are antibiotic-resistant bacteria or auxotrophic bacteria.

[0018] In some of these embodiments, the antibiotic-resistant bacteria is ampicillin-resistant Escherichia coli.

[0019] In some embodiments, the culture temperature is 36° C.-37.8° C., and the bacteria having antibiotic resistance are cultured in an air environment for 16 h-20 h.

[0020] In some of these embodiments, the screening agent is an antibiotic, or a nutrient-deficient culture medium, or a bacterial metabolite.

[0021] In some embodiments, the screening agent is an antibiotic, and the screened bacteria is Escherichia coli. Preferably, the concentration of the antibiotic in the mixed droplets is 4-6 ug / mL, and further, the concentration of the antibiotic in the mixed droplets is 4.5-5.5 ug / mL.

[0022] In some embodiments, step S1 includes: injecting the diluted bacterial solution into the digital microfluidic chip to form bacterial solution droplets with an electrode base of 2 to the power of N, and based on the volume of a single electrode droplet being 3 nL, the amount of bacteria in each bacterial solution droplet is 18-22 bacteria per 64 electrode bases, and there is at least one bacterial solution droplet; N is a positive integer equal to or greater than 1.

[0023] Further preferably, the separation and acquisition of a single bacterial droplet in step S1 comprises the following steps:

[0024] S1.1 Cultivate bacteria in culture medium and dilute the bacterial solution with culture medium;

[0025] S1.2 injecting the diluted bacterial solution onto the digital microfluidic chip to form bacterial solution droplets with 2 to the power of N as the electrode base number, the amount of bacteria in each bacterial solution droplet is 18-22 bacteria per 64 electrode bases, and the bacterial solution droplet is at least 1; N is a positive integer equal to or greater than 1;

[0026] S1.3. By controlling the power on and off of the electrodes on the chip, the droplets can be manipulated and controlled to move in opposite directions. The droplets are torn in half to obtain droplets that have been torn in half. According to the above method, the droplets that have been torn in half are torn in half several times to form 2 to the power of N single-electrode droplets. S1.4. The single-electrode droplets are detected and judged for the number of bacteria through the identification system. Droplets containing only one bacterium are marked, and the electrode containing the droplet containing only one bacterium is continuously powered to keep the droplet in place, thus obtaining a single-bacteria droplet.

[0027] In some embodiments, the Nth power of 2, N is 1-8, preferably 5-6.

[0028] In some of the embodiments, in S1.3, the amount of bacteria in each bacterial liquid droplet is 19-21 bacteria per 64 electrode bases.

[0029] In some embodiments, based on a single electrode droplet volume of 3 nL, in step S2, the bacterial concentration after dilution is 70-120 cfu / uL, preferably 90-110 cfu / uL. Or when the bacteria are cultured to an OD600 of about 1.5-2.5, the culture is stopped and the bacterial solution is diluted with a culture medium to an OD600 of 0.2-0.4.

[0030] In some embodiments, the antibiotic is ampicillin, or ampicillin and kanamycin.

[0031] In some embodiments, the following steps are also included:

[0032] S1.5 For droplets that do not contain bacteria or contain more than one bacterium, the movement of the droplets is controlled by controlling the power on and off of the electrodes on the chip, and the empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and they are re-merged into an independent empty droplet that does not contain bacteria and a droplet with multiple bacteria, respectively. The empty droplets are removed and the droplets with multiple bacteria are recovered.

[0033] In some embodiments, the separation method further comprises:

[0034] S1.6 controls the power on and off of the electrodes on the chip to move the droplets containing a single bacterium and rearrange them into a droplet matrix with one electrode between them;

[0035] After S1.7 recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment, and the multiple recovered bacterial droplets are merged into a new droplet with 2N as the electrode base, and the above steps S1.3-S1.7 are repeated until the recovered droplets contain no bacteria, and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria. After the present invention uses an efficient single bacteria separation method based on a digital microfluidic system, the separated single bacteria are cultured on a digital microfluidic platform to achieve the cultivation of multiple separated single bacteria, such as Escherichia coli (facultative anaerobic bacteria), Bacillus congestus, Bacillus subtilis (aerobic bacteria), Bifidobacterium infantis, and Lactobacillus casei (anaerobic bacteria).

[0036] The single bacteria screening and continuous culture method based on the digital microfluidic system of the present invention has the following advantages: after obtaining single bacteria droplets in a simple, efficient and high-throughput manner, screening is performed, and the separated single bacteria droplets can be continuously cultured in situ on the chip without transferring them outside the chip, so that bacteria can be multiplied, ensuring that the obtained single bacteria will not be unable to grow due to other reasons such as transfer, which ultimately leads to sample loss. Moreover, through continuous in situ culture, the contamination rate is low, which greatly saves costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the structure of a digital microfluidic chip.

[0038] Figure 2 It is a schematic diagram of the effect of magnetic beads with different luminescence intensities in Example 2, wherein A is the negative control group, and BF are different single bacteria isolated.

[0039] Figure 3 In Example 3, the protein expression in the droplets after the isolated single bacteria were cultured was observed under a fluorescence microscope, wherein A is the wild-type intern-GFP, which does not emit green light when observed under a high-power microscope without the addition of an inducer, and B is the wild-type intern-GFP, which emits green light when observed under a high-power microscope when an inducer is added.

[0040] Figure 4 It is a schematic diagram of the results of bacterial screening and continuous culture in Example 7.

[0041] Explanation of the reference numerals: 10, upper electrode plate, 20, lower electrode plate, 101, fluid channel layer, 201, microdroplets, 21, hydrophobic layer, 22, dielectric layer, 23, electrode layer, 24, electrode, 11, upper cover, 12, dielectric layer, 13, hydrophobic layer. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0045] In some embodiments of the present invention, taking Escherichia coli as an example, a single bacteria screening and culturing method based on digital microfluidics is provided, comprising the following steps:

[0046] 1. Depending on the type of bacteria, dilute the bacteria to an OD600 of about 0.2-0.4 using appropriate culture medium, and perform fluorescent staining on the bacterial solution as needed;

[0047] 2. After staining, continue to dilute it by an appropriate multiple (1000-2000 times) to make the bacterial concentration 50-100 cfu / uL;

[0048] 3. In the digital microfluidic system, the bacterial solution is injected into the chip, and the droplets are manipulated by controlling the power on and off of the electrodes on the chip; the droplets will move toward the adjacent powered electrode and away from the powered-off electrode; in this way, the droplets of equal volume are controlled to move in the opposite direction, split in half, and quickly form single-electrode droplets;

[0049] 4. Use the fluorescence detection system to mark the droplets containing only one bacterium, and move and rearrange the marked droplets; recover the droplets without bacteria or containing more than one bacteria through the preset path program, and repeat the above steps until the recovered droplets contain no bacteria and the empty liquid is discharged;

[0050] 5. Control the chip temperature through the temperature control system and select the temperature suitable for bacterial growth;

[0051] 6. According to the bacteria's demand for oxygen, choose whether to start the anaerobic culture system; if it is aerobic bacteria or facultative anaerobic bacteria, there is no need to control the gas environment; if it is oxygen-tolerant anaerobic bacteria, the anaerobic culture system can be started, and nitrogen should be continuously passed through the environment for 10 minutes in advance to adjust the environment to an anaerobic state;

[0052] 7. If specific screening of bacteria is required, the corresponding reagents such as antibiotics can be injected. After the single droplets are separated, they are mixed with the bacterial solution to obtain mixed droplets. The concentration of antibiotics in the mixed droplets is 4-6ug / mL.

[0053] After the culture is completed, if further experiments are required on the successfully cultured bacterial solution, such as enzyme activity, promoter effect detection, etc., appropriate reagents are injected for subsequent experimental operations according to specific needs.

[0054] The study found that the concentration of the screening agent (such as antibiotics) for ampicillin-resistant strains was significantly different from the concentration in the conventional method (100ug / mL), and it was also found that the concentration of antibiotics had a significant impact on bacterial screening in single bacterial droplets in the digital microfluidics system. Only an appropriate concentration can screen ampicillin-resistant strains and culture them for growth.

[0055] The screening and culturing method of the present invention can integrate the processes of single bacteria isolation, screening and culturing on a chip, with a high degree of automation, simple operation and low risk of introducing contamination. It is not necessary to transfer the isolated and cultured bacteria outside the chip for screening and identification, and the isolated and cultured bacteria can be directly screened and identified on the digital microfluidic platform.

[0056] It is clear to those skilled in the art that the steps included in the culture method of the present invention do not need to be limited to a specific order, and those skilled in the art can make appropriate adjustments to the order based on their common sense.

[0057] In order to better understand the present invention, the digital microfluidic platform used in the following embodiments is introduced: a panoramic camera is configured to observe the overall operation of the chip and droplets; a microscopic camera is used to observe the contents (such as magnetic beads) in the droplets; a micro-optical measurement module is used to measure the OD of bacteria on the chip 600 Absorbance; Magnetic adsorption module, which can be programmed according to the preset and automatically move to the area where magnetic adsorption is required with 3 axes; Chip structure, the sandwich structure of the chip currently used is displayed; Temperature control module, used to control the temperature of the chip area; Electric three-axis control platform, used to digitally control the movement of the chip platform under different lenses. For details, see Figure 1 : The digital microfluidic chip includes an upper electrode plate 10 and a lower electrode plate 20, and a fluid channel layer 101 is formed between the upper electrode plate 10 and the lower electrode plate 20; the lower electrode plate 20 includes an electrode layer 23, and the electrode layer 23 includes a plurality of electrodes arranged in an array.

[0058] In the following embodiments, the power-on condition (light-proof) of the digital microfluidic chip used is:

[0059] Line voltage: 45V,

[0060] Column voltage: 40V,

[0061] Holding time: 10um,

[0062] Frame delay time: 1ms,

[0063] The row and column voltages are set to a lower voltage overnight to ensure the performance of the chip the next day.

[0064] Interval power on: Power on and off alternately at the final resting position of the single bacterial droplet, 1000ms on, 1000ms off.

[0065] Temperature control: Maintain at 37°C overnight.

[0066] The present invention is further described in detail below with reference to specific embodiments.

[0067] The present invention is further described in detail below with reference to specific embodiments.

[0068] Example 1

[0069] The single bacteria screening and culturing method based on the digital microfluidics system described in this embodiment comprises the following steps:

[0070] 1. Use LB medium containing 100ug / mL ampicillin to culture ampicillin-resistant Escherichia coli to an OD600 of about 2.0. Use LB medium to culture wild-type Escherichia coli to an OD600 of about 2.0.

[0071] 2. Use LB medium to dilute the ampicillin-resistant Escherichia coli and wild-type Escherichia coli to an OD600 of about 0.2.

[0072] 3. Dilute the two bacterial solutions with OD600 values ​​of about 0.2 by 1000 times with LB medium to make the bacterial concentration about 70-120 cfu / uL.

[0073] The two bacterial solutions were injected into a chip with a single electrode droplet volume of 3 nL to form a droplet with 64 grids (2 to the sixth power of the electrode base) to which the wild-type intein-GFP and mutant intein-GFP were distributed.

[0074] There are 20 intein-GFP Escherichia coli in total; by controlling the power on and off of the electrodes on the chip, the droplets are manipulated, and the droplets move toward the adjacent powered electrode and away from the powered-off electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction, split in half, and quickly form single-electrode droplets. The droplet array is fluorescently detected and judged through the fluorescence imaging recognition system, and the droplets containing only one bacterium are marked, and 20 single-bacteria droplets are obtained;

[0075] 4. Separating and obtaining a droplet containing a single ampicillin-resistant Escherichia coli and a single wild-type Escherichia coli;

[0076] 5. Inject different concentrations of ampicillin on the chip to separate single-electrode ampicillin droplets, control the antibiotic droplets of different concentrations, and mix them with droplets of Escherichia coli containing ampicillin resistance and droplets of wild-type Escherichia coli, respectively. The final concentration of ampicillin in the mixed droplets of ampicillin droplets and ampicillin-resistant Escherichia coli or wild-type Escherichia coli is 2.5-10ug / mL, as shown in the following table;

[0077] 6. The temperature of the chip was set to 37°C through the temperature control system, and the E. coli was cultured in an air environment for 18 hours to observe the growth of the E. coli in the droplets.

[0078] Table 1. Statistics of E. coli growth status

[0079]

[0080] When the concentrations were 4, 5, and 6 ug / mL, the antibiotics were screened as critical concentrations. The results showed that the bacteria grew the most at 4 ug / mL and the least at 6 ug / mL. In general, the growth was best at 5 ug / mL.

[0081] When the concentration of the antibiotic in the mixed droplets is 4-6 ug / mL, Amp-resistant Escherichia coli can be well screened.

[0082] Example 2

[0083] The single bacteria screening and continuous culture method based on the digital microfluidic system described in this embodiment includes the following steps:

[0084] 1. Using phage display technology on the chip, the phage antibody library was screened for three rounds, and the E. coli produced after the screening was diluted 800-1000 times with LB culture medium (containing 0.1% F68) on the chip to make the bacterial concentration about 70cfu / uL-90cfu / uL; the diluted bacterial solution was injected into a chip with a total number of 16384 electrodes, and each chip formed 64 groups of 64 grids (2 to the sixth power of the electrode base) of droplets, and the volume of each single electrode droplet was 3nL. On average, each group was allocated 20 bacteria per 64 electrode bases;

[0085] 2. The droplets are manipulated by controlling the power on and off of the electrodes on the chip. The droplets will move toward the adjacent powered electrode and away from the powered-off electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction and are torn in half with equal volume, so that each group obtains two droplets after being torn in half. According to the above method, the droplets torn in half are torn in half again for a second round to obtain four droplets, and then the four droplets are torn in half for a third round to obtain eight droplets. A total of six rounds of half-tear are performed to quickly form 64 groups of 64 single-electrode droplets, and droplets containing a single Escherichia coli are separated. The droplet array is subjected to fluorescence detection and judgment through a fluorescence imaging recognition system, and droplets containing only one bacterium are marked. The electrode where the droplet is located is continuously powered to keep the droplet in place. Among the 64 groups, 16-19 single bacterial droplets are basically separated, and an average of about 18 single bacterial droplets are separated in each group, with a separation rate of 90.0%. The success rate of single bacterial separation in 64 electrodes is 28.1%. The experiment found that when the number of bacteria assigned to each group was less than 18 or greater than 22, the separation rate and separation success rate would be greatly reduced. The separation results can be seen in Table 2.

[0086] Table 2

[0087]

[0088]

[0089] For droplets without bacteria or with more than one bacteria, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and re-merged into an independent empty droplet without bacteria and a droplet with multiple bacteria, respectively, and the empty droplet is removed and the droplet with multiple bacteria is recovered;

[0090] By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a droplet matrix with one electrode between them.

[0091] After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet with 2 to the power of N as the electrode base. The above steps are repeated until the recovered droplets contain no bacteria and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria.

[0092] 3. Inject helper phage M13K07 (titer: 10 11 pfu / mL), and the two were torn in half with equal volumes to separate the single-electrode assisted phage droplets, and the assisted phage droplets and the E. coli droplets were controlled to mix;

[0093] 4. Use the temperature control system to set the chip to 37°C and incubate for 2 hours to allow the helper phage to infect E. coli.

[0094] 5. Inject 10ug / mL of ampicillin and kanamycin mixed antibiotics onto the chip, separate and obtain 1*2 electrode droplets, control the antibiotic droplets to mix with the E. coli droplets infected by the auxiliary phage, and the concentration of the mixed antibiotics of ampicillin and kanamycin in the mixed droplets is 5ug / mL;

[0095] 6. Then, the E. coli was cultured in air for 18 hours, and the successfully grown droplets were marked on the software; the ungrown droplets were removed from the chip, the temperature was adjusted to 30°C, and cultured for 3 hours to produce more phage progeny;

[0096] 7. Inject antigen-coupled carboxyl magnetic beads (20 μm) onto the chip, separate droplets containing single magnetic beads, control the mixing of single magnetic bead droplets and grown E. coli droplets, and incubate at room temperature for 2 h;

[0097] 8. Inject M13-PE antibody (1:1000, Sino Biological) containing 0.1% F68 onto the chip, separate and obtain single droplets, mix the control antibody droplets and the E. coli droplets producing phage progeny to obtain antigen magnetic beads)-(phage)-(M13-PE), and incubate at room temperature for 30 minutes;

[0098] 9. Use a fluorescence microscope to detect whether the magnetic beads in the droplets emit fluorescence and the intensity of the fluorescence to determine the affinity differences between different clones;

[0099] 10. Control the droplets through electrodes and output the droplets you want to obtain (e.g., with higher fluorescence intensity) for cultivation.

[0100] See also Figure 2 , A is the negative control group, which theoretically does not fluoresce. BF is the different single bacteria separated, and the phages produced by them show different antibodies. The affinity between antibodies and antigens is different, which ultimately makes the magnetic beads capture phages differently in the same time, and finally manifests as the difference in fluorescence intensity.

[0101] The more phages are bound to the magnetic beads, the higher the fluorescence intensity of the phage dye is. Figure 2 The fluorescence intensity of B, C, and D is higher.

[0102] Example 3

[0103] The single bacteria screening and continuous culture method based on the digital microfluidic system described in this embodiment includes the following steps:

[0104] 1. Use LB medium containing 100ug / mL ampicillin to culture the mixed bacterial liquid of wild-type intein-GFP and mutant intein-GFP Escherichia coli until OD600 is about 2.0;

[0105] 2. Dilute the mixed bacterial solution with LB medium to an OD600 of about 0.2;

[0106] 3. Dilute the mixed bacterial solution with an OD600 value of about 0.2 with LB medium 1000 times to make the bacterial concentration about 70-100 cfu / uL;

[0107] 4. Inject the diluted bacterial solution into a chip with a single-electrode droplet volume of 3 nL to form a droplet with 64 grids (2 to the sixth power of the electrode base), and the droplet is allocated to 20 wild-type intein-GFP and mutant intein-GFP Escherichia coli in total;

[0108] 5. According to Example 1 or Example 2, the control of the droplets is achieved by controlling the power on and power off of the electrodes on the chip; the droplets will move toward the adjacent powered electrode and away from the powered-off electrode; in this way, the equal-volume droplets are controlled to move in the opposite direction, and the equal-volume droplets are torn in half to quickly form single-electrode droplets, and the single-electrode droplets are detected and judged, and the droplets containing only one bacterium are marked, and finally 20 single-bacteria droplets are obtained;

[0109] 6. Inject 10ug / mL of ampicillin on the chip, control the antibiotic droplets to mix with the separated single electrode droplets (single bacteria droplets), and the concentration of the mixed antibiotics in the mixed droplets is 5ug / mL; 7. Set the chip to 37°C through the temperature control system, and culture E. coli in air environment for 18h;

[0110] 8. After the culture is completed, add 1*2 grid LB medium containing 10uM IPTG, and form a single electrode culture medium droplet by controlling the power on and off of the electrode on the digital microfluidic chip. Mix it with the E. coli cultured with resistance, and induce protein expression at 37°C for 3h;

[0111] 9. The protein expression was then observed under a fluorescence microscope. Under FITC excitation light, it was observed that the E. coli in some droplets emitted green light. The genotypes of the E. coli in the droplets that emitted green light and those in the droplets that did not emit green light were analyzed by sequencing, corresponding to the wild-type intein-GFP E. coli and the mutant intein-GFP E. coli, respectively.

[0112] from Figure 3It can be seen that under the 10x eyepiece and 40x objective lens, the wild-type intern-GFP does not emit green light without the addition of inducers, and no luminescence is observed under a high-power microscope, and the single bacteria separation is 100% successful. When the wild-type intern-GFP is added with inducers, it emits green light under a high-power microscope, and the single bacteria grow well after culture.

[0113] Example 4

[0114] In this embodiment, the crowded Corynebacterium is isolated and cultured, comprising the following steps:

[0115] 1. Cultivate the crowded bacterium in a medium containing BHI + Tween 80 (concentration 0.0625%) until OD600 is about 1.0.

[0116] 2. Use BHI+Tween 80 (concentration 0.0625%) culture medium to dilute the crowded bacterium to an OD600 of 0.2-0.3.

[0117] 3. Dilute the bacterial solution with an OD600 value of about 0.2-0.3 by 1000 times with BHI+Tween 80 (concentration 0.0625%) culture medium to a bacterial concentration of about 70-100 cfu / uL;

[0118] The bacterial solution was injected into a chip with a single electrode droplet volume of 3 nL, forming a droplet with 64 grids (2 to the sixth power of the electrode base), and 20 bacteria were distributed to the droplet;

[0119] 4. According to the method of Example 1 or the embodiment, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and the droplets will move toward the adjacent powered electrode and away from the powered-off electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction, split in half, and quickly form single-electrode droplets, separating 19 to droplets containing a single crowded bacterium;

[0120] For droplets without bacteria or with more than one bacteria, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and re-merged into an independent empty droplet without bacteria and a droplet with multiple bacteria, respectively, and the empty droplet is removed and the droplet with multiple bacteria is recovered;

[0121] By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a droplet matrix with one electrode between them.

[0122] After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet with 2 to the power of N as the electrode base. The above steps are repeated until the recovered droplets contain no bacteria and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria.

[0123] 5. The temperature of the chip was set at 37°C through the temperature control system, and the crowded Corynebacterium was cultured in an air environment for 20 hours. After the culture, it was clearly observed that the droplets were obviously turbid, and under a microscope, it was observed that the droplets were full of crowded Corynebacterium.

[0124] Example 5

[0125] In this embodiment, Bacillus subtilis is isolated and cultured, comprising the following steps:

[0126] 1. Cultivate Bacillus subtilis in NB broth until OD600 is about 2.0;

[0127] 2. Use NB broth medium to dilute Bacillus subtilis to an OD600 of 0.15-0.25;

[0128] 3. Dilute the bacterial solution with an OD600 value of about 0.15-0.25 by 1000 times with NB broth medium to make the bacterial concentration about 80-120 cfu / uL;

[0129] 4. The bacterial liquid is injected into a chip with a single-electrode droplet volume of 3nL to form a droplet of 64 grids (2 to the sixth power of the electrode base). The droplet is assigned to 20 bacteria. The droplets are manipulated by controlling the power on and off of the electrodes on the chip in the manner of Example 1 or Example 2. The droplets will move toward the adjacent powered electrode and away from the powered-off electrode. In this way, droplets of equal volume are controlled to move in the opposite direction and are torn in half to quickly form a single-electrode droplet, and droplets containing 18 single crowded rod bacteria are separated;

[0130] For droplets without bacteria or with more than one bacteria, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and re-merged into an independent empty droplet without bacteria and a droplet with multiple bacteria, respectively, and the empty droplet is removed and the droplet with multiple bacteria is recovered;

[0131] By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium can be moved again.

[0132] Rearrange into a droplet matrix with one electrode between them;

[0133] After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet with 2 to the power of N as the electrode base. The above steps are repeated until the recovered droplets contain no bacteria and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria.

[0134] 5. Then inject the NB broth culture medium into the digital microfluidic chip, and form a single-electrode culture medium droplet by controlling the power on and off of the electrodes on the digital microfluidic chip, and mix it with the screened bacterial droplets to continue culturing;

[0135] 6. The temperature of the chip was set at 37°C through the temperature control system, and Bacillus subtilis was cultured in air environment for 16 hours. After the culture, it was obviously observed that the droplets were obviously turbid, and under the microscope, it was observed that the droplets were full of Bacillus subtilis.

[0136] Example 7

[0137] The single bacteria screening and continuous culture method based on the digital microfluidic system described in this embodiment includes the following steps:

[0138] 1. Use LB medium containing 50ug / mL ampicillin to transfer E. coli containing the nanoantibody gene for transforming anti-GFP protein at a ratio of 1:50; the bacterial concentration after dilution with LB medium is 11-14.5cfu / uL; 2. Inject the bacterial solution into a chip with a single electrode droplet volume of 25nL to form a 16-grid droplet, which is distributed to 5 bacteria and cultured on the chip at 37°C for 1h;

[0139] 3. According to Example 1 or Example 2, the electrodes on the control chip are powered on and off to control the droplets; the 16-grid bacterial solution is torn to tear, and 2 grids of droplets without bacteria are torn out and discarded, and 2 grids of LB culture medium containing 5ug / mL ampicillin are controlled by electrodes to mix with the remaining 14 grids of bacterial solution and culture at 37°C; the liquid replacement operation is repeated every 15 minutes to achieve continuous perfusion culture, replace the antibiotic concentration in the droplets, and continue 4 times for a total of 1 hour;

[0140] 4. According to Example 1 or Example 2, the electrodes on the chip are powered on and off to control the droplets; 80uM IPTG (isopropyl-β-D-thiogalactoside) solution is injected into two electrodes on the chip and mixed with the bacterial solution to a final concentration of 10uM;

[0141] 5. According to Example 1 or Example 2, the electrodes on the chip are controlled to be powered on and off to control the droplets; 18 grids of bacterial solution are controlled to be torn, 2 grids of droplets are torn out and discarded, and 2 grids of LB culture medium containing 5ug / mL ampicillin and 100uM IPTG are controlled by electrodes to be mixed with the remaining 16 grids of bacterial solution and cultured at 37°C. The liquid replacement operation is repeated every 15 minutes to achieve continuous perfusion culture, and the concentration of antibiotics and inducers is maintained by perfusion culture;

[0142] 6. According to Example 1 or Example 2, the control of the droplets is achieved by controlling the power on and off of the electrodes on the chip; after continuous perfusion culture for 1 hour, 18 grids of bacterial liquid are controlled to be torn every 15 minutes, 2 grids of droplets are torn out for detection and standby, and the remaining droplets continue to maintain perfusion culture;

[0143] 7. Control the droplets through electrodes, add 2 droplets containing GFP protein-coupled magnetic beads to the 2 droplets torn out in step 6, and incubate at 37°C for 1 hour;

[0144] 8. After the incubation, the magnetic beads are adsorbed by a magnet, and the electrodes are controlled to wash the magnetic beads in the droplets by injecting new droplets containing PBS buffer and tearing off the old droplets;

[0145] 9. After washing, add Anti-Flag-Cy3 and incubate at 37°C for 1 hour; then observe the protein expression under a fluorescence microscope, and judge the time when E. coli begins to produce the target nanoantibody and the target nanoantibody concentration reaches the best based on the fluorescence intensity of the magnetic beads under 546nm excitation light.

[0146] See also Figure 4 ,in, Figure 4 A is the first sample added 1h 30min after perfusion culture. The magnetic beads emit weak fluorescence, and the secreted antibody reaches the detection limit. Figure 4 BG is a sample of every 15 minutes, and the fluorescence continues to increase. The fluorescence intensity of H is consistent with that of G, indicating that the concentration of secreted antibodies has reached its peak. This method can be easily operated and efficiently achieve the effect of single bacterial screening and in situ continuous culture.

[0147] The single bacteria screening and continuous culture method based on the digital microfluidic system of the present invention can greatly reduce the probability of contamination. For example, the probability of contamination for each culture is about 1%. The traditional operation is to culture continuously for 10 times, and the probability of contamination = 1% + [(100% - 1%) * 1%], and the contamination rate is shown in Table 3, 0.099. However, the separation and continuous culture method of the present invention, even if the continuous culture is 10 times, the probability of contamination is about 1%. Automated screening depends on the chip area. If A1A is taken as an example, the flux is about 1000. Taking A3B4 as an example, the flux of the prior art is about 3000, and the theoretical flux is close to 10000. Compared with the traditional method, the difference can reach 1000 times. Compared with some reagents with relatively high costs, the cost saving is more than 1000 times.

[0148] Table 3. Contamination rate of 10 consecutive cultures using the traditional method

[0149]

[0150] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A single bacteria screening and cultivation method based on a digital microfluidics system, characterized in that: The single bacteria screening and culturing method is carried out on a digital microfluidic chip, which includes an upper electrode plate and a lower electrode plate, wherein a fluid channel layer is formed between the upper electrode plate and the lower electrode plate; the lower electrode plate includes an electrode layer, wherein the electrode layer includes a plurality of electrodes arranged in an array; The single bacteria screening and culturing method comprises the following steps: S1. Inject the diluted bacterial solution into the digital microfluidic chip, and tear the electrode on the digital microfluidic chip into half with equal volume by controlling the power on and off to form a single electrode droplet. Detect and judge the single electrode droplet, mark the droplet containing only one bacterium, and obtain a single bacteria droplet; S2. Injecting the screening material onto the chip of the digital microfluidic system, separating and obtaining single-electrode screening material droplets, controlling the single-electrode screening material droplets, and mixing them with the single bacterial droplets to obtain mixed droplets; S3. Setting the culture temperature, culturing the bacteria in the mixed droplets, and screening the bacteria; S4. Inject the culture medium into the digital microfluidic chip, and form single-electrode culture medium droplets by controlling the power on and off of the electrodes on the digital microfluidic chip. Mix the culture medium droplets with the screened bacterial droplets and continue culturing.

2. The single bacteria screening and culturing method according to claim 1, characterized in that: The following steps are also included: S5. Continue culturing for a period of time. After the bacteria have multiplied for at least one generation, add a droplet of new culture medium, and tear the bacterial droplet into half of equal volume by controlling the power on and off of the electrodes on the digital microfluidic chip to obtain a bacterial droplet injected with the new culture medium; S6. Repeat step S5 for more than 0 times to achieve continuous perfusion culture.

3. The single bacteria screening and culturing method according to claim 1, characterized in that: The bacteria are bacteria containing antibiotic resistance or auxotrophic bacteria, or bacteria capable of producing certain metabolites.

4. The single bacteria screening and culturing method according to claim 1, characterized in that: The screening agent is an antibiotic, or a nutrient-deficient culture medium, or a bacterial metabolite.

5. The single bacteria screening and culturing method according to claim 4, characterized in that: The screening object is an antibiotic, and the screened bacteria is Escherichia coli. Preferably, the concentration of the antibiotic in the mixed droplet is 4-6 ug / mL, and more preferably 4.5-5.5 ug / mL.

6. The single bacteria screening and culturing method according to any one of claims 1 to 4, characterized in that: Step S1 includes: injecting the diluted bacterial solution onto the digital microfluidic chip to form bacterial solution droplets with 2 to the power of N as the electrode base, and based on the volume of a single electrode droplet being 3 nL, the amount of bacteria in each bacterial solution droplet is 18-22 bacteria per 64 electrode bases, and the number of bacterial solution droplets is at least 1; N is a positive integer equal to or greater than 1.

7. The single bacteria screening and culturing method according to claim 6, characterized in that: The S1 step includes the following steps: S1.1 Cultivate bacteria in culture medium and dilute the bacterial solution with culture medium; S1.2 injecting the diluted bacterial solution onto the digital microfluidic chip to form bacterial solution droplets with 2 to the power of N as the electrode base number, the amount of bacteria in each bacterial solution droplet is 18-22 bacteria per 64 electrode bases, and the bacterial solution droplet is at least 1; N is a positive integer equal to or greater than 1; S1.3 controls the power on and off of the electrodes on the chip to achieve control of the droplets, controls droplets of equal volume to move in opposite directions, and tears the droplets in half to obtain droplets that have been torn in half; repeats the tearing in half several times for the droplets that have been torn in half in the above manner to form 2 to the power of N single-electrode droplets; S1.4 detects and determines the number of bacteria in the single-electrode droplets through the identification system, marks droplets that contain only one bacterium, and continuously supplies power to the electrode where the droplet that contains only one bacterium is located to keep the droplet in place, thus obtaining a single-bacteria droplet.

8. The single bacteria screening and culturing method according to claim 7, characterized in that: The Nth power of 2, N is 1-8, preferably 5-6.

9. The single bacteria screening and culturing method according to claim 7, characterized in that: In S1.2, the amount of bacteria in each bacterial liquid droplet is 19-21 bacteria per 64 electrode bases.

10. The single bacteria screening and culturing method according to claim 7, characterized in that: In step S1.1, the bacterial concentration after dilution is 70-120 cfu / uL, preferably 90-110 cfu / uL.

11. The single bacteria screening and culturing method according to claim 7, characterized in that: The following steps are also included: S1.5 For droplets that do not contain bacteria or contain more than one bacterium, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and re-merged into an independent empty droplet that does not contain bacteria and a droplet with multiple bacteria, respectively. The empty droplet is removed and the droplet with multiple bacteria is recovered.

12. The single bacteria screening and culturing method according to claim 11, characterized in that: The separation method further comprises: S1.6 controls the power on and off of the electrodes on the chip to move the droplets containing a single bacterium and rearrange them into a droplet matrix with one electrode between them; After S1.7 is recovered, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet with 2 to the power of N as the electrode base. The above steps S1.3-S1.7 are repeated until the recovered droplets contain no bacteria and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria.

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