阅读说明：本技术 一种使用液滴微流控芯片的高通量筛选方法在放线菌中的应用 (Application of high-throughput screening method using droplet microfluidic chip in actinomycetes ) 是由 涂然 王猛 张玥 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种使用液滴微流控芯片的高通量筛选方法在放线菌中的应用,通过本方法可以将放线菌单孢子被包埋到单液滴中,在液滴中0-7d的培养时间内间稳定成型保证了宽泛的检测及分选时间,在检测方面,用此方法可以高通量检测到在放线菌中有功能的启动子,或鉴定一些未知启动子的强度,并筛选到适宜强度的启动子元件用于后续的放线菌菌株改造；在筛选方面,此方法的筛选通量可以达到10～5个菌株/天,可以对产生绿色荧光信号的阳性菌和不产生绿色荧光信号的阴性菌的混库进行成功分选,其中分选后的阳性菌富集率达到81.7％以上,相较分选前的3.1％提高至26倍。(The invention discloses an application of a high-throughput screening method using a droplet microfluidic chip in actinomycetes, through the method, single spores of the actinomycetes can be embedded into a single droplet, the stable forming is carried out within the culture time of 0-7d in the droplet, the wide detection and sorting time is ensured, in the aspect of detection, promoters which have functions in the actinomycetes can be detected in a high-throughput manner by the method, or the strength of some unknown promoters can be identified, and promoter elements with proper strength are screened for subsequent modification of actinomycetes strains; in the aspect of screening, the screening flux of the method can reach 10 5 The strains per day can be successfully sorted from the mixed library of the positive bacteria generating green fluorescent signals and the negative bacteria not generating green fluorescent signals, wherein the enrichment rate of the sorted positive bacteria reaches more than 81.7 percent and is improved to 26 times compared with 3.1 percent before sorting.)

1. The application of the high-throughput screening method using the droplet microfluidic chip in actinomycetes is characterized by comprising the following steps of:

step one, using a liquid culture medium to separate actinomycete spores from a solid platePurging, filtering to remove mycelia adhered to spores until the spores in the filtrate are monodisperse, and diluting spore suspension with sterile liquid culture medium to concentration of 0.5-2 × 10⁶Per mL;

embedding the spores and the culture medium obtained in the step one into a liquid drop by adopting a liquid drop microfluidic technology, wherein the diameter of the liquid drop is 80-100 mu m;

step three, performing static culture on the liquid drops until the fluorescent signals in the liquid drops can be detected by a fluorescent microscope, and finishing the culture before the mycelial clusters generated by the germination of actinomycetes spores are full of all the liquid drops;

step four, analyzing and sorting the droplets by utilizing droplet microfluidic detection screening equipment according to the intensity of the fluorescence signal in the droplets, and screening out one or more droplets with relatively high fluorescence intensity;

and step five, carrying out pure culture and verification on spores in the sorted liquid drops.

2. The application of the high-throughput screening method using the droplet microfluidic chip according to claim 1 to actinomycetes, wherein in the step one, the liquid culture medium is R2YE liquid culture medium.

3. The application of the high-throughput screening method using the droplet microfluidic chip in actinomycetes, according to claim 1, wherein in the second step, the ratio of the oil phase to the water phase flow rate is in the range of 1:1 to 3:1, and the prepared droplet size is 80-100 μm.

4. The use of the high throughput screening method using droplet microfluidic chips according to claim 3 in actinomycetes, wherein the size of the droplet is determined by the following procedure:

embedding the spores and the culture medium obtained in the first step into a liquid drop by adopting a liquid drop microfluidic technology, adjusting the flow rate to obtain liquid drops with different sizes, and observing and measuring under a microscope to determine the size of the liquid drop.

5. The use of the high-throughput screening method using a droplet microfluidic chip according to claim 1 in actinomycetes, wherein the temperature of the culture is 30 ℃; the duration of the culture is preferably 18-24 h.

6. The use of the high throughput screening method using droplet microfluidic chip of claim 5 in actinomycetes, wherein the duration of the culture is determined by the following procedure:

and D, performing culture in a determination stage by adopting a droplet microfluidic technology to obtain droplets in the second step, observing the germination condition and the fluorescent signal condition of spores in the droplets, determining that the fluorescent signal of the droplets can be observed under a fluorescent microscope, and finishing the culture before all the droplets are filled with mycelial clusters generated by germination of hyphae generated by the germination of the spores.

7. The use of the method of claim 1 for high throughput screening using droplet microfluidic chips on actinomycetes, wherein said actinomycetes is Streptomyces lividans.

Technical Field

The invention belongs to the technical field of microorganism biology. More specifically, the invention relates to the application of a high-throughput screening method using a droplet microfluidic chip in actinomycetes.

Background

Actinomycetales belongs to the class of Actinomycetes and is a high GC, filamentous growth gram-positive prokaryote which propagates mainly as asexual spores. The order of Actinomycetales includes many genera including Actinomycetes and Streptomyces, but there are great similarities between genera in terms of life habits, morphological development and propagation patterns, for example: most of the plants are saprophytic; during solid plate culture, developed mycelia can be mostly differentiated, and the mycelia are divided into basal mycelia responsible for absorbing nutrition and aerial mycelia extending to the surrounding space; the aerial hyphae become spore hyphae after being mature, and the spore hyphae are further differentiated into monospores for propagation; during liquid culture, hyphae intertwine with each other to form a hypha mass.

The secondary metabolic processes of actinomycetes can produce a variety of important secondary metabolites, such as antibiotics, hydrolases, enzyme inhibitors, immunomodulators, pigments, and the like. Of the antibiotics currently found, about half are produced by actinomycetes. Therefore, the research and the modification of the actinomycetes have important significance for exploring new natural products and producing valuable natural products by taking the actinomycetes as a chassis. However, whether the transformation is guided by metabolites or protein expression, a powerful high-throughput screening method is necessary as a support, and the ideal phenotype can be efficiently screened from thousands or even millions of strain banks in a short time. The current high-throughput screening method aiming at the actinomycetes is very limited, which greatly limits the research and the modification of the actinomycetes.

The droplet microfluidic technology is a new high-throughput screening technology developed in recent years, and has the advantages that a culture medium in which single cells are suspended can be embedded in droplets, the droplets are suspended in an oil phase and do not interfere with each other, and each droplet can be used as an independent microreactor to culture cells, express proteins and produce metabolites. And then, the microchip is used for analyzing and sorting the detection of the substance signals in the liquid drops to form a technical platform comprising cell culture, signal detection and separation and sorting, and the method has the advantages of high flux, less consumables, high survival rate, high positive rate and the like. At present, droplet microfluidics has been successfully applied to high-throughput screening of various bacteria and fungi, but no report of applying droplet microfluidics technology to high-throughput screening and detection of actinomycetes exists at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an application of a high-throughput screening method using a droplet microfluidic chip in actinomycetes.

The invention is realized by the following technical scheme:

the application of the high-throughput screening method using the droplet microfluidic chip in actinomycetes is carried out according to the following steps:

step one, washing actinomycete spores from a solid plate by using a liquid culture medium, filtering mycelium adhered to the spores until the spores in the filtrate are in a monodispersed state, and diluting a spore suspension by using the sterile liquid culture medium until the concentration is 0.5-2 multiplied by 10⁶Per mL;

embedding the spores and the culture medium obtained in the step one into a liquid drop by adopting a liquid drop microfluidic technology, wherein the diameter of the liquid drop is 80-100 mu m;

step three, performing static culture on the liquid drops until the fluorescent signals in the liquid drops can be detected by a fluorescent microscope, and finishing the culture before the mycelial clusters generated by the germination of actinomycetes spores are full of all the liquid drops;

step four, analyzing and sorting the droplets by utilizing droplet microfluidic detection screening equipment according to the intensity of the fluorescence signal in the droplets, and screening out one or more droplets with relatively high fluorescence intensity;

and step five, carrying out pure culture and verification on spores in the sorted liquid drops.

Preferably, in the application of the high-throughput screening method using the droplet microfluidic chip to actinomycetes, in the step one, the liquid culture medium includes, but is not limited to, R2YE liquid culture medium, and also includes other liquid culture media capable of germinating actinomycetes spores, including YEME, R2, R5, 2 × YT, LB liquid culture medium, and the like.

Preferably, in the application of the high-throughput screening method using the droplet microfluidic chip in actinomycetes, in the second step, the ratio of the flow speed of the oil phase to the flow speed of the water phase is in the range of 1:1 to 3:1, and the size of the prepared droplet is 80-100 μm.

Preferably, in the application of the high-throughput screening method using the droplet microfluidic chip to actinomycetes, in the second step, the size of the droplet is determined by the following process:

embedding the spores and the culture medium obtained in the first step into a liquid drop by adopting a liquid drop microfluidic technology, adjusting the flow rate to obtain liquid drops with different sizes, and observing and measuring under a microscope to determine the size of the liquid drop.

Preferably, in the application of the high-throughput screening method using the droplet microfluidic chip in actinomycetes, in the third step, the temperature of the culture is 30 ℃; the duration of the culture is preferably 18-24 h.

Preferably, in the application of the high-throughput screening method using the droplet microfluidic chip to actinomycetes, in the third step, the duration of the culture is determined by the following process:

and D, performing culture in a determination stage by adopting a droplet microfluidic technology to obtain droplets in the second step, observing the germination condition and the fluorescent signal condition of spores in the droplets, determining that the fluorescent signal of the droplets can be observed under a fluorescent microscope, and finishing the culture before all the droplets are filled with mycelial clusters generated by germination of hyphae generated by the germination of the spores.

Preferably, the high-throughput screening method using the droplet microfluidic chip is applied to actinomycetes, which are in the order of actinomycetes, including but not limited to actinomycetes and streptomyces, all of which are propagated by monospores and can be proliferated and divided by the monospores and differentiated into microorganisms in the form of hyphae. The starting strain used in the present invention is one of the actinomycete model strains, and belongs to Streptomyces lividans of Streptomyces of Actinomycetales.

The invention has the advantages and beneficial effects that:

1. in the method, a single spore is embedded into a single liquid drop, and a liquid drop is used as a reaction micro unit to perform a series of physiological activities and biochemical reactions such as protein expression, production of secondary metabolites and the like. The vigorous expression of protein is mostly in the vegetative growth stage (0-3d) of the growth and development initial stage of actinomycete cells, and the production of secondary metabolites is mostly in the secondary metabolism stage (4-7d) of the middle and later stages of the growth and development of the cells, and experiments prove that the droplets are not punctured by soft mycelial clusters formed by the differentiation of single spores of the actinomycete within the culture time of 0-7d in the droplets, and the long-time stable forming of the droplets ensures relatively wide detection and sorting time, so that the detection which takes protein signals as targets in the initial stage and the detection which takes the secondary metabolites as targets in the middle and later stages can be smoothly realized.

2. Different from escherichia coli and bacillus subtilis which are used for constructing a screening system in the prior art, the individual size of actinomycetes and the growth characteristics of a formed mycelium pellet have unique individuality, and how to adjust the technical parameters such as suspension concentration of the actinomycetes and the size of liquid drops enables the growth and detection of the actinomycetes to be suitable for the liquid drop microfluidic chip screening system is the core technical problem solved by the invention; experiments prove that when the ratio of the spore concentration to the number of the liquid drops is between 0.3 and 0.5, and the diameter of the prepared liquid drop is between 80 and 100 mu m, the forming ratio and the detection speed of the spore liquid drop are optimal. E.g. spore suspension to a concentration of 1X 10⁶The number of droplets per mL is 2-3.7X 10⁶The spore liquid drop with high efficiency can be prepared by every mL, and the diameter of the liquid drop is 80-100 μm. The diameter of the liquid drop is 80-100 μm, on one hand, the stability of the liquid drop is ensured in the whole growth and secondary metabolism stage of actinomycetes, and on the other hand, the generated fluorescence can adapt to the rapid detection and screening of the liquid drop microfluidic chip.

3. The invention adopts the droplet microfluidic technology to carry out high-throughput detection and screening on actinomycetes. In the aspect of detection, the method can detect promoters which are functional in actinomycetes in high throughput, or identify the strength of some unknown promoters, and screen promoter elements with proper strength for subsequent modification of actinomycetes strains; in the aspect of screening, the screening flux of the method can reach 10⁵Compared with the speed of hundreds of strains per day in a common pore plate and a shake flask, the detection and screening flux of the invention is improved by nearly thousand times per day. And successfully sorting the mixed library of the positive bacteria generating the green fluorescence signals and the negative bacteria not generating the green fluorescence signals, wherein the enrichment rate of the sorted positive bacteria reaches over 81.7 percent and is improved to 26 times compared with that of the positive bacteria before sorting by 3.1 percent.

Drawings

FIG. 1 is a flow chart of the application of a high throughput screening method using a droplet microfluidic chip to actinomycetes in an embodiment of the present invention;

FIG. 2 is a microscopic view of monodisperse spores according to a second embodiment of the present invention;

FIG. 3-A is a schematic flow diagram of a droplet generation process using a microfluidic chip;

FIG. 3-B is a diagram showing the state of droplets in the oil phase and the water phase in different proportions;

FIG. 3-C is a statistical plot of droplet sizes for oil phase and water phase at different ratios;

FIG. 4-A is a diagram showing the germination state of spores subjected to ordinary standing culture at culture times of 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h in the fourth example of the present invention;

FIG. 4-B is a diagram showing the spore germination state in the liquid drop at the culture time of 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h in the fourth embodiment of the invention;

FIG. 4-C is a graph showing the spore status in the droplet and the fluorescence signal of the droplet at 12h, 18h, 24h, 36h, 2d, 3d, 6d and 7d incubation times in example four of the present invention;

FIG. 5 is a graph showing the comparison of the fluorescence signals of the droplets of the negative and positive strains at the time of detection and sorting (24h) in example five of the present invention;

FIG. 6 is a graph of the fluorescent signal intensity of droplets from different promoter strains according to example six of the present invention;

FIG. 7 is a graph showing the detection results of green fluorescence signal values of the hollow droplets and the yin-yang strains in example seven of the present invention.

For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.

Detailed Description

In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.

The model actinomycete starting strain for high-throughput screening by the droplet microfluidic technology referred to in the following examples is Streptomyces lividans (Streptomyces lividans) 66.

Example construction of strains that are negative and positive for Green fluorescent Signal

Based on the original enhanced green fluorescent protein gene egfp (as shown in SEQ NO.1 in the sequence table), ATG is used as an initiation codon, and all GTG codons in an expression frame are synonymously mutated into GTC or GTA, so that egfp (ATG) gene (as shown in SEQ NO.2 in the sequence table) is obtained. Based on egfp (ATG), the initial ATG is replaced by ACG by using a point mutation method to obtain the egfp (ACG) gene (as shown in SEQ NO.3 in a sequence table). 5 heterologous promoters were selected (table 1): p_gapdh(EL)，P_rpsL(XC)，P_rpsL(SG)，P_rpsL(TP)，P_erm*ESynthesizing the promoter sequences, and respectively cloning the promoters by taking the synthesized sequences as templates; simultaneously, 4 endogenous promoters were selected (table 1): p_rpS12，P_gpmA，P_pyk，P_rpoAAnd cloning the promoters by taking the genome of the streptomyces lividans as a template. These 9 promoters were ligated in front of the egfp (ATG) gene, respectively, and the fusion fragment of "promoter + gene" was ligated to the hygromycin-resistant integration vector pSET152-hyg, to construct 9 green fluorescent protein expression plasmids:

pSET152-hyg-P_gapdh(EL)-egfp (ATG), wherein P_gapdh(EL)The sequence of the promoter is shown as SEQ NO.4 in the sequence table;

pSET152-hyg-P_rpsL(XC)-egfp (ATG), wherein P_rpsL(XC)The sequence of the promoter is shown as SEQ NO.5 in the sequence table;

pSET152-hyg-P_rpsL(SG)-egfp (ATG), wherein P_rpsL(SG)The sequence of the promoter is shown as SEQ NO.6 in the sequence table;

pSET152-hyg-P_rpsL(TP)-egfp (ATG), wherein P_rpsL(TP)The sequence of the promoter is shown as SEQ NO.7 in the sequence table;

pSET152-hyg-P_erm*E-egfp (ATG), wherein P_erm*EThe sequence of the promoter is shown as SEQ NO.8 in the sequence table;

pSET152-hyg-P_rps12-egfp (ATG), wherein P_rps12The sequence of the promoter is shown as SEQ NO.9 in the sequence table;

pSET152-hyg-P_gpmA-egfp (ATG), wherein P_gpmAThe sequence of the promoter is shown as SEQ NO.10 in the sequence table;

pSET152-hyg-P_pyk-egfp (ATG), wherein P_pykThe sequence of the promoter is shown as SEQ NO.11 in the sequence table;

pSET152-hyg-P_rpoA-egfp (ATG), wherein P_rpoAThe sequence of the promoter is shown as SEQ NO.12 in the sequence table;

at the same time, P is added_rpsL(XC)The promoter was ligated before the egfp (ACG) gene and the fusion fragment of "promoter + gene" was ligated to the hygromycin-resistant integrative vector pSET152-hyg, constructing an inactivated green fluorescent protein expression plasmid: pSET152-hyg-P_rpsL(XC)Egfp (ACG). The promoter sources used for the above plasmids, the plasmids and strains constructed therefrom are shown in Table 1 below, and the information on the primers used therein is shown in Table 2 below.

TABLE 1 promoter sources and plasmids and strains constructed

Table 2 primers used in the examples

The 10 plasmids constructed above are respectively transformed into the protoplast of the original strain streptomyces lividans by a PEG-mediated protoplast transformation method, and the protoplast is coated on an R2YE solid plate. After incubation at 30 ℃ for 20h, a final concentration of 50. mu.g was addedThe transformant can be obtained by screening hygromycin/mL and further culturing at 30 ℃ for 5 days. Transformants were selected for re-passaging on the same concentration of hygromycin resistant R2YE plates, yielding 9 green fluorescent signal positive strains with the egfp (ATG) gene integrated: lividans/pSET152-hyg-P_gapdh(EL)-egfp(ATG)，S. lividans/pSET152-hyg-P_rpsL(XC)-egfp (ATG) (SLATG for short), S. lividans/pSET152-hyg-P_rpsL(SG)-egfp(ATG)，S.lividans/pSET152-hyg-P_rpsL(TP)-egfp(ATG)， S.lividans/pSET152-hyg-P_erm*E-egfp(ATG)，S.lividans/pSET152-hyg-P_rpS12-egfp(ATG)，S. lividans/pSET152-hyg-P_gpmA-egfp(ATG)，S.lividans/pSET152-hyg-P_pyk-egfp(ATG)，S. lividans/pSET152-hyg-P_rpoA-egfp (ATG), and green fluorescence signal negative strain S.lividans/pSET152-hyg-PrpsL (XC) -egfp (ACG) (SLACG) integrated with egfp (ACG) gene.

EXAMPLE two plate spore culture and Collection

Spores (20% glycerol solution) frozen at-80 deg.C were suspended, and a small amount of spore liquid was dipped with a sterile inoculating loop and streaked on an R2YE solid plate for subculture. The plate was placed upside down in an incubator at 30 ℃ for 5-7 days until gray mature spores were differentiated. For collection, 5mL of sterile R2YE liquid medium that had been passed through a 0.22 μm aqueous membrane was added to the surface of the plate, and spores on the plate were repeatedly aspirated by a pipette and suspended in the medium. 3mL of the eluted spore suspension was added to a sterile 8-layer filter device and filtered 2 times until the spores were monodisperse in the medium (as shown in FIG. 2). And (3) dripping 20-30 mu L of the filtered spore suspension on a blood counting chamber, and observing and counting the spore concentration under a 20X objective lens. The spore suspension was diluted to a concentration of 1X 10 with sterile R2YE liquid medium⁶one/mL.

EXAMPLE three droplet embedding method

The droplets were subjected to embedding according to the methods in the publications He, R., Ding, R., Heyman, J.A.et al.J Ind Microbiol Biotechnol (2019)46:1603.https:// doi.org/10.1007/s 10295-019-: wherein the water phase is 1mL and the spore concentration is 1X 10⁶Spore suspension of one/mL, mixing the aboveThe aqueous phase was added to a 1mL syringe and the oil phase was 1mL of stabilizer-containing oil, which was also added to the 1mL syringe, and then the syringe containing the aqueous phase and the oil phase was communicated to the droplet generation microchip in an attempt to adjust the ratio of the flow rates of the oil phase and the aqueous phase to 1:1, 2:1 and 3:1 to create droplets of different diameters (fig. 3-B, C). When the ratio of the spore concentration to the number of the droplets is between 0.3 and 0.5 and the diameter of the prepared droplets is between 80 and 100 mu m, the formation ratio of the spore droplets and the detection speed are optimal. The size of the prepared droplets is dependent on the chip and the flow rate, e.g. spore suspension to a concentration of 1X 10⁶The number of droplets per mL is 2-3.7X 10⁶The spore liquid drop with high efficiency can be prepared by every mL, and the diameter of the spore liquid drop is 80-100 mu m. It was found that droplets having a diameter of 90 μm were most suitably produced when the flow rates of the oil phase and the water phase were 2:1, respectively, and the flow rates of the oil phase and the water phase were 1000. mu.L/h and 500. mu.L/h, respectively. After the liquid drops are stably generated, the liquid drops are inoculated into a sterile 1.5mL centrifuge tube and are subjected to static culture at 30 ℃ for detection; and (4) inoculating the liquid drops into a 1mL syringe if the liquid drops need to be sorted, and performing static culture at 30 ℃.

Example four observations of hyphal germination and droplet stability in droplets

The spore suspension obtained in the first example was subjected to static culture at 30 ℃, and a part of the bacterial solution was taken at different times (2h, 4h, 6h, 8h, 10h, 12h, 24h) and mounted on a slide for sectioning and observed under a microscope 20 Xobjective (microscope: Leica DM 5000B). Meanwhile, the liquid drops obtained in the third example are placed at 30 ℃ for static culture, and part of the liquid drops are taken at different times (2h, 4h, 6h, 8h, 10h, 12h, 18h, 24h, 36h, 2d, 3d, 6d, 7d) for observation, and the specific operation is as follows: aspirate 3-5 μ L of the drop into a hemocytometer and aspirate 15-20 μ L of the drop embedding oil into the hemocytometer so that the oil phase fills the entire hemocytometer and is viewed under a microscope 20 Xobjective. And (3) comparison finding: the spores can normally germinate and differentiate in the liquid drops; within 24h, the germination of spores in the droplets was the same as under normal conditions (FIG. 4-A, B); after 1d, up to 7d, the mycelial pellets, although substantially filling the droplets, did not puncture the droplets, and the droplets remained stable (fig. 4-C), providing a basis for elastic selection of sorting time.

EXAMPLE observation of Green fluorescence Signal in five droplets

Observing the fluorescent signal of the liquid drop obtained in the third example under different culture time, and specifically operating as follows: and sucking 3-5 mu L of the liquid drop prepared in the third example and placing the liquid drop in a blood counting plate, sucking 15-20 mu L of liquid drop embedding oil in the blood counting plate to ensure that the oil phase is filled in the whole blood counting plate, and placing the sample to be subjected to microscopic examination prepared in the third example under a 20x objective lens to observe the fluorescence condition of the liquid drop. The exposure time of the white field is 30ms, the fluorescence condition of the liquid drop is simultaneously observed under the same visual field, the fluorescence excitation wavelength is 488nm, the absorption wavelength is 520nm, and the fluorescence field is observed and shot under the exposure time of 10ms, 30ms and 50ms respectively. In 12h to 7d, the normal germination and differentiation of spores in the liquid drop can be observed under the condition of static culture of the liquid drop, and the fluorescent signal in the liquid drop can be detected by a fluorescent microscope; the optimal detection and sorting time in the future experiment was determined to be 24h (fig. 4-C) as the fluorescence signal intensity in 24h of droplet was optimal based on the fluorescence signal intensity of the droplet at different times. At the time of detection and sorting (24h), a significant difference was observed in the fluorescent signals of the strains with various promoters of different intensities (FIG. 5). And the fourth embodiment has verified that the droplet can be kept stable at this time, so the detection and sorting conditions are met.

Example six droplet microfluidic assay to identify different promoter strengths

At the sorting time (24h), a green fluorescent signal positive strain S.lividans/pSET152-hyg-P, which is embedded with 9 different promoters constructed in example one, is used_gapdh(EL)-egfp(ATG)，S. lividans/pSET152-hyg-P_rpsL(XC)-egfp(ATG)，S.lividans/pSET152-hyg-P_rpsL(SG)-egfp(ATG)，S. lividans/pSET152-hyg-P_rpsL(TP)-egfp(ATG)，S.lividans/pSET152-hyg-P_erm*E-egfp(ATG)，S. lividans/pSET152-hyg-P_rpS12-egfp(ATG)，S.lividans/pSET152-hyg-P_gpmA-egfp(ATG)，S. lividans/pSET152-hyg-P_pyk-egfp(ATG)，S.lividans/pSET152-hyg-P_rpoA-egfp (ATG) -droplets were separately detected for green fluorescence and the strain S. livi was negative for green fluorescence signaldans/pSET152-hyg-P_rpsL(XC)Egfp (ATG) as control. The intensities of the fluorescence signals of the individual strains were plotted to give an ordering of the different promoter intensities (FIG. 6). By using the method, promoters which are functional in actinomycetes can be detected in high throughput, or the strength of some unknown promoters can be identified, and promoter elements with proper strength can be screened for subsequent actinomycetes strain modification.

Example seven-droplet microfluidic sorting Green fluorescent Signal negative and positive bacteria mixing library

The drop obtained in example three was incubated at 30 ℃ for 24 h. Before sorting, the fluorescence signal values of the empty droplet, the green fluorescence signal negative strain SLACG and the green fluorescence signal positive strain SLATG are respectively detected (figures 7A, B and C), and the result shows that the difference between the fluorescence signal values of the empty droplet and the SLACG is small, but the fluorescence signal value of the SLATG is obviously improved compared with the fluorescence signal values of the empty droplet and the SLACG, so that the sorting condition is achieved. During sorting, the flow rate of the droplets is set to be 20 mu L/h, the flow rate of the oil phase is set to be 100 mu L/h, the screening speed of the droplets is 10-15/s, the sorting threshold value is 1% of the highest cell signal of the droplets, the deflection voltage is 700V, and about 40 droplets are collected in a 1.5mL sterile centrifuge tube each time.

Example eight validation of the sorting strains

The drops collected by sorting in example seven were spread on R2YE plates containing 50. mu.g/mL hygromycin and cultured at 30 ℃ for 3 days to grow single colonies. And then, placing the flat plate under blue light for irradiation, respectively counting the number of positive colonies emitting green fluorescence and the number of negative colonies not emitting green fluorescence on the flat plate, and calculating the positive enrichment rate. The positive enrichment rate is calculated by the formula: positive enrichment (%). times.100% for the number of positive colonies/(number of positive colonies + number of negative colonies). Through calculation, in the invention, the enrichment rate of the green fluorescence signal positive bacteria can be increased from 3.1% of the initial premixing to 81.7%, and the positive enrichment rate is increased to 26 times of the original rate, as shown in the following table.

TABLE 3 statistics of positive enrichment rate for microfluidic sorting of droplets

While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

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aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 2

<211> 720

<212> DNA

<213> unknown ()

<400> 2

atggtcagca agggcgagga gctgttcacc ggggtcgtac ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtc tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtacc ctggcccacc 180

ctcgtcacca ccctgaccta cggcgtccag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtca agttcgaggg cgacaccctg 360

gtcaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tcaacttcaa gatccgccac aacatcgagg acggcagcgt ccagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tcctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtaaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 3

<211> 671

<212> DNA

<213> unknown ()

<400> 3

tcgagctgga cggcgacgta aacggccaca agttcagcgt ctccggcgag ggcgagggcg 60

atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtac 120

cctggcccac cctcgtcacc accctgacct acggcgtcca gtgcttcagc cgctaccccg 180

accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 240

gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtc aagttcgagg 300

gcgacaccct ggtcaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 360

tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 420

agcagaagaa cggcatcaag gtcaacttca agatccgcca caacatcgag gacggcagcg 480

tccagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtcctgctgc 540

ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 600

atcacatggt cctgctggag ttcgtaaccg ccgccgggat cactctcggc atggacgagc 660

tgtacaagta a 671

<210> 4

<211> 285

<212> DNA

<213> unknown ()

<400> 4

gctgctcctt cggtcggacg tgcgtctacg ggcaccttac cgcagccgtc ggctgtgcga 60

cacggacgga tcgggcgaac tggccgatgc tgggagaagc gcgctgctgt acggcgcgca 120

ccgggtgcgg agcccctcgg cgagcggtgt gaaacttctg tgaatggcct gttcggttgc 180

tttttttata cggctgccag ataaggcttg cagcatctgg gcggctaccg ctatgatcgg 240

ggcgttcctg caattcttag tgcgagtatc tgaaagggga tacgc 285

<210> 5

<211> 302

<212> DNA

<213> unknown ()

<400> 5

gccctgcagg cggaagtcag gtagacacga cttccgctag tccttgcaag gtctgctgac 60

gtgaggcggg gcggtcgttt ttgaccgccc tgccttcgtc atgtaggctc gctcgctgtg 120

cctggcgtgt catcagacgc ccaggtcccg gtgccgtgag gcccgggcca tcgagccggt 180

ggtacgtggc tgcggtcccc ttgtgagggc tgcgcgccgt gtgctgtccg gcgcgcacag 240

ccttgaatcc acccgcgggg gccggccggt ctccgtgagc tcgagtagac gacggagacg 300

ta 302

<210> 6

<211> 311

<212> DNA

<213> unknown ()

<400> 6

aggggcgcgc ggccccggcc gccggccgtc ccgtccgggg gcccgtggcc ggggcggtcc 60

cggcgtgtcg cggcggacag catttgtttt gacccagctc cgtgaggtag gtacgctcaa 120

gccttgtgcc tggggtgtgc ctgggctcgg gtgcgtgtcc tcaaccgcat cgcgagtccg 180

tcagtagcca ccgcaatctg cgcccttcct gccttcgggg cgggagtccg cagtattcga 240

cacacccgac cgcgtgggtc ggcgatgttc caggttagtt tcacgaacgg cacacagaaa 300

ccggagaagt a 311

<210> 7

<211> 363

<212> DNA

<213> unknown ()

<400> 7

accgggtccg cgatcggcgg aggcgaacac ttttgaccac tatgagttca tccaggtaag 60

cttggtcgcc gtgcctggtg aatgccagga cgatcgctcg tgcccatact gcaggccgga 120

cctccgggac cacgtacgga cacgcgacac gcccgacctc ggggtacgtg cgaggcgggt 180

agctacttcc cggaaacggg actgaccagc agagacggcg aaagccggaa ctgccggtgg 240

ggccgcgctg cggaagtcgc accctcgatg agggacggct tgcccggagc tacaaccgca 300

gcagtagagc caccggccgg acggccgatg gcagccgaaa cgaagtaagg aacctgcgct 360

tct 363

<210> 8

<211> 282

<212> DNA

<213> unknown ()

<400> 8

ggtaccagcc cgacccgagc acgcgccggc acgcctggtc gatgtcggac cggagttcga 60

ggtacgcggc ttgcaggtcc aggaagggga cgtccatgcg agtgtccgtt cgagtggcgg 120

cttgcgcccg atgctagtcg cggttgatcg gcgatcgcag gtgcacgcgg tcgatcttga 180

cggctggcga gaggtgcggg gaggatctga ccgacgcggt ccacacgtgg caccgcgatg 240

ctgttgtggg ctggacaatc gtgccggttg gtaggacacc ac 282

<210> 9

<211> 282

<212> DNA

<213> unknown ()

<400> 9

ctcgccgaac aggacaaagt gggatagagc gggcggcgat gccggtgtcg cccatttgtt 60

ttgaccgcag cgaatgcgct aggtacgctc ataccttgtg cctggggtgt gccctggccc 120

tcgtgcgtgt ctacagccgc accgggggcc gtgtgtggcc accgcatttc gcgtctcctt 180

ccgcctcgcg gcgggagttc gcggcttcga cacacccgac cgcgtgggtc ggtgacgttc 240

caggttagct tcaccattcg gcacacagaa accggagaag ta 282

<210> 10

<211> 300

<212> DNA

<213> unknown ()

<400> 10

tggtcaggta cagcgccatg aaggtggcga cgaaggcacc gagccggttg accagggtgc 60

tggtccacag ccaccagaac gcgcggggga gcccggagac ggtctcgcgt gcggcacgtc 120

cgagtccggc gacaggcatg ggtcccccga ggtgattgag atcgccgtaa gcggctgatg 180

cggtaggcac aacttacaaa cggctctctc ggggaagcca tccaattaac acttcccgtc 240

aaccgtccgc ccaccgggcg cacgcgcggg ggatcagggc cttggattac gctcggaagc 300

<210> 11

<211> 299

<212> DNA

<213> unknown ()

<400> 11

ggggagaact cggcacccgt gcgcgaggcg gccgtggccg gactggaggg gctcggcctg 60

gcggtcgacg gcgggctgaa cgccgtacgc ggcgacggag cccggctgat ctcgcccgcg 120

ggggcgcggg tggcggtggc ggtggtaccg acggacgagg aaatggagat cgcgacacag 180

acctacgcgc tggtaaatga atcggggaat cccgatctca cctgagcggc atcccgccct 240

tttgtatttt ccgccagacg gaatattccg cgccgaaaca aaccgatagg atggcaccc 299

<210> 12

<211> 287

<212> DNA

<213> unknown ()

<400> 12

atgcgcaagg tcgacgtctt cgtcaagggc ccgggttccg gtcgtgagac cgccatccgc 60

tccctgcagg cgaccggcct cgaggtcggc tccatccagg acgtcacgcc caccccgcac 120

aacgggtgcc gtccgccgaa gcgtcgtcgc gtctgatccc gcttcaccgc ggcatcgctt 180

caccagggtt ttccgggcgg tacggctctt tcgggtcgta tcgcccgtac ccttgcagta 240

ctggtcgggc gtcaaatagc gggcgcccct gactgaagga tcaccac 287