Biosensor for screening high-yield apramycin strains based on fluorescence signals

Abstract

The invention constructs a biosensor which responds to the apramycin concentration and generates obvious fluorescence signal change, and provides a biosensor skeleton which can be adapted to other antibiotics. Specifically, the invention relates to the expression cassette comprising the apramycin resistance gene sequence, the apramycin resistance gene sequence comprising the expression cassette, a method for preparing the apramycin biosensor, and application of the apramycin biosensor to detection of a bacterial strain for producing apramycin. The method accelerates the step of screening the high-yield apramycin strain, provides a biosensor skeleton for screening other antibiotics, and potentially accelerates the screening of the antibiotic high-yield strain with the corresponding resistance gene.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of screening high-yield apramycin strains, in particular to a biosensor for screening high-yield apramycin strains, and more particularly to a biosensor for screening high-yield apramycin strains based on fluorescence signals. BACKGROUND

[0003] Currently, the industrialization of apramycin faces three major challenges. The first challenge is the lack of detection sensitivity: traditional methods (such as HPLC) are costly, time-consuming, and difficult to quickly quantify trace residues. The second challenge is the difficulty of production quality control: the activity of the strain fluctuates greatly during fermentation, affecting the stability of the yield. The third challenge is the low efficiency of screening: traditional plate screening has a high false positive rate, slowing down the breeding process of high-yield strains. Therefore, it is urgent to develop an efficient detection method to realize rapid determination of apramycin to solve the problem of apramycin industrial production bottleneck. The biosensor based on fluorescence signal can feedback the content change of intracellular target substance in real time, and produce measurable signal.

[0004] Therefore, it is necessary to develop a biosensor based on fluorescence signal to screen high-yield apramycin strains, and to have a rapid real-time response apramycin high-throughput detection method, which has a profound significance for the practical application of apramycin. SUMMARY

[0005] As can be seen, the prior art about obtaining high-yield apramycin strains still has the above limitations. The present application aims to break through these limitations and seek a solution to high-throughput screening of high-yield apramycin strains based on a biosensor based on fluorescence signals. In view of these limitations, the problems that the prior art needs to solve at present mainly include: (1) constructing a biosensor that can respond to apramycin; (2) building a high-throughput screening mixed bacteria system to improve work efficiency.

[0006] In order to solve the problems in the prior art, the present application first constructs an expression cassette containing apramycin resistance gene sequence, and then uses the expression cassette to construct a biosensor that can quickly and real-time respond to apramycin, which lays a relatively solid foundation for solving the problem of rapid acquisition of high-yield apramycin strains. One object of the present application is to construct a biosensor that can respond to apramycin. Another object of the present application is to assemble a biosensor that can respond to succinic acid. For this purpose, the present application constructs a biosensor that responds to the concentration of apramycin and produces a significant change in fluorescence signal, and provides a biosensor skeleton that can be adapted to other antibiotics.

[0007] Specifically, the present application adopts the following technical solutions:

[0008] Item 1. An expression cassette, wherein the expression cassette comprises an apramycin resistance gene sequence, a promoter sequence, and a replicon sequence.

[0009] Item 2. The expression cassette of item 1, wherein the apramycin resistance gene sequence is set forth in SEQ ID NO: 1.

[0010] Item 3. The expression cassette of item 1 or 2, wherein the promoter sequence is selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NO: 2, a nucleotide sequence set forth in SEQ ID NO: 3, a nucleotide sequence set forth in SEQ ID NO: 4, and a nucleotide sequence set forth in SEQ ID NO: 5.

[0011] Item 4. The expression cassette of item 1 or 2, wherein the promoter sequence is set forth in SEQ ID NO: 3.

[0012] Item 5. The expression cassette of any one of items 1 to 4, wherein the replicon sequence is selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NO: 6, a nucleotide sequence set forth in SEQ ID NO: 7, and a nucleotide sequence set forth in SEQ ID NO: 8.

[0013] Item 6. The expression cassette of any one of items 1 to 4, wherein the replicon sequence is set forth in SEQ ID NO: 7.

[0014] Item 7. An apramycin biosensor comprising a plasmid vector comprising the expression cassette of any one of items 1 to 6, preferably the apramycin biosensor further comprises a fluorescent gene, further preferably the apramycin biosensor is Escherichia coli.

[0015] Item 8. A method for preparing an apramycin biosensor, comprising the steps of:

[0016] providing a fluorescent Escherichia coli without resistance;

[0017] constructing a plasmid vector comprising the expression cassette of any one of items 1 to 6;

[0018] introducing the plasmid vector into the fluorescent Escherichia coli without resistance to obtain a fluorescent Escherichia coli competent cell as an apramycin biosensor.

[0019] Item 9. Use of an apramycin biosensor for detecting the concentration of apramycin, wherein the apramycin biosensor is the apramycin biosensor of item 7.

[0020] Item 10. The use according to Item 9, characterized in that the apramycin biosensor is placed in a strain that produces apramycin, the fluorescence intensity produced by the apramycin biosensor is measured, and the apramycin biosensor is determined to meet the requirements based on the fluorescence intensity data. Attached Figure Description

[0021] Figure 1 The results of promoter screening during the construction and optimization of the expression cassette are shown, illustrating the fluorescence change trend of the biosensor under the regulation of promoters with different expression intensities. Figure 1 A is in P rpsU Fluorescence intensity of biosensors under the regulation of promoters; Figure 1 B is in P glpD Fluorescence intensity of biosensors under the regulation of promoters; Figure 1 C is in P rplY Fluorescence intensity of biosensors under the regulation of promoters; Figure 1 D is in P cspA The fluorescence intensity of a biosensor is regulated by a promoter.

[0022] Figure 2 The results of replicon screening during the construction and optimization of the expression cassette are shown, and the fluorescence change trend of the biosensor is displayed under different copy number regulation.

[0023] Figure 3 This demonstrates the response range of the apramycin biosensor of this application to apramycin concentration.

[0024] Figure 4 This demonstrates the fluorescence intensity produced by the apramycin biosensor of this application in response to different concentrations of different antibiotics.

[0025] Figure 5 The image shows a microscopic examination of the droplets generated by the droplet single-cell sorting system (DREM cell) of this application using the apramycin biosensor, wherein: Figure 5 A shows the bright-field view of the droplet. Figure 5 B shows the dark field view of the droplets.

[0026] Figure 6 The diagram shows the real-time signal and sorting signal of the droplets generated by the droplet single-cell sorting system (DREM cell) of this application using the apramycin biosensor, wherein: Figure 6 A is a real-time signal graph of the DREM cell droplet; Figure 6 B is a scatter plot of the DREM cell droplet signal; Figure 6 C is the histogram of droplet signals in the DREM cell. Detailed Implementation

[0027] The exemplary embodiments of this application are described herein with reference to the drawings, in which are shown by way of illustration specific details of certain embodiments of this application; it is not intended that the application be limited to these embodiments. It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described and illustrated herein without departing from the spirit and scope of the application. Thus, it is intended that the present application cover the modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

[0028] The terms used in this application, unless otherwise indicated, have their ordinary meanings in the art.

[0029] As used herein, the term "biosensor" refers to an analytical device that integrates a biological element (such as an enzyme, antibody, cell, etc.) with a physical or chemical transducer (such as an electrode, optical element, etc.). It is capable of converting information generated by a biological recognition process into a quantifiable electrical signal or other form of signal output, thereby enabling the detection and analysis of specific biomolecules, cells, or biological processes.

[0030] As used herein, the term "nucleotide" or "nucleic acid" means a deoxyribonucleotide, deoxyribonucleoside, ribonucleoside, or ribonucleotide, in either single or double stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides, which analogues have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically indicated otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, simplifying codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Additionally, "gene" as used herein is also intended to mean the same.

[0031] As used herein, the term "vector" is used to describe a nucleic acid molecule that can be engineered to contain a polynucleotide or polynucleotides that can be replicated in a host cell. Vectors include, but are not limited to, nucleic acid molecules which are single-stranded, double-stranded or partially double-stranded; nucleic acid molecules which are circular, linear, or partially linear; nucleic acid molecules which comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." A recombinant expression vector of the application can comprise a nucleic acid of the application in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector includes one or more regulatory elements that can be selected based upon the host cell used to express the nucleic acid sequence to be expressed.

[0032] In one aspect, the present application provides an expression cassette, wherein the expression cassette comprises an apramycin resistance gene sequence, a promoter sequence, and a replicon sequence. The expression cassette of the present application can be constructed by screening promoters and replicons and combining them with the apramycin resistance gene.

[0033] In one embodiment of the expression cassette, the apramycin resistance gene sequence is set forth in SEQ ID NO: 1.

[0034] In one embodiment of the expression cassette, the promoter sequence is selected from the group consisting of a nucleotide sequence set forth in SEQ ID NO: 2, a nucleotide sequence set forth in SEQ ID NO: 3, a nucleotide sequence set forth in SEQ ID NO: 4, and a nucleotide sequence set forth in SEQ ID NO: 5. In another embodiment of the expression cassette, the promoter sequence is set forth in SEQ ID NO: 2. In another embodiment of the expression cassette, the promoter sequence is set forth in SEQ ID NO: 3. In another embodiment of the expression cassette, the promoter sequence is set forth in SEQ ID NO: 4. In another embodiment of the expression cassette, the promoter sequence is set forth in SEQ ID NO: 5.

[0035] In one embodiment of the expression cassette, the replicon sequence is selected from the group consisting of: a nucleotide sequence as set forth in SEQ ID NO: 6, a nucleotide sequence as set forth in SEQ ID NO: 7, and a nucleotide sequence as set forth in SEQ ID NO: 8. In another embodiment of the expression cassette, the replicon sequence is as set forth in SEQ ID NO: 6. In another embodiment of the expression cassette, the replicon sequence is as set forth in SEQ ID NO: 7. In another embodiment of the expression cassette, the replicon sequence is as set forth in SEQ ID NO: 8.

[0036] In another aspect, the present application provides an apramycin biosensor. The apramycin biosensor of the present application contains a plasmid vector comprising the expression cassette of the present application.

[0037] In a preferred embodiment, the apramycin biosensor of the present application further contains a fluorescent gene. In a further preferred embodiment, the apramycin biosensor of the present application is E. coli.

[0038] In another aspect, the present application provides a method for preparing an apramycin biosensor, comprising the steps of: providing a fluorescent E. coli without resistance; constructing a plasmid vector comprising the expression cassette of the present application; introducing the plasmid vector into the fluorescent E. coli without resistance to obtain a fluorescent E. coli competent cell as an apramycin biosensor.

[0039] In another aspect, the present application provides the use of an apramycin biosensor for detecting the concentration of apramycin, wherein the apramycin biosensor is the apramycin biosensor of the present application described above. In a preferred embodiment, the use is characterized in that the apramycin biosensor of the present application described above is put into a strain producing apramycin, the fluorescence intensity generated by the apramycin biosensor is determined, and whether the apramycin biosensor meets the requirements is determined according to the fluorescence intensity data.

[0040] The present application constructs a biosensor that responds to the concentration of apramycin and produces a significant change in fluorescence signal, and provides a biosensor skeleton that can be adapted to other antibiotics. Moreover, the present application constructs a biosensor that responds to apramycin based on the apramycin resistance gene, which has stable response between batches and has broad application prospects in the screening of high-yield apramycin strains. The present application accelerates the pace of screening high-yield apramycin strains, and provides a biosensor skeleton for screening other antibiotics, which potentially accelerates the screening of high-yield strains of antibiotics with corresponding resistance genes.

[0041] EXAMPLE

[0042] The application will be further described below with reference to the drawings and specific examples so that those skilled in the art can better understand the application and implement it. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0043] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0044] Example 1: Preparation of fluorescent E. coli competent cells

[0045] After activating the non-resistance gene carrying E. coli, it was transferred to a 50 mL centrifuge tube placed in an ice bath and kept for 10 minutes. After centrifuging at 4°C and 200 rpm for 5 minutes, the supernatant was discarded. The bacterial cells were resuspended with 2.5 mL of pre-cooled 0.1 mol / L CaCl2. After centrifuging at 4°C and 200 rpm for 5 minutes, the supernatant was discarded. The bacterial cells were gently resuspended with 1 mL of pre-cooled 0.1 mol / L CaCl2(containing 15% glycerol). The resulting non-resistance gene carrying E. coli competent cells were aliquoted and stored at -80°C.

[0046] Example 2: Construction and optimization of amikacin biosensor

[0047] Example 2.1: Construction and optimization of promoters

[0048] The apramycin resistance gene as shown in SEQ ID NO. 1 (synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.) was placed under the control of four different promoters P glpD (sequence as shown in SEQ ID NO: 2, synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.), P cspA (sequence as shown in SEQ ID NO: 3, synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.), P rplY (sequence as shown in SEQ ID NO: 4, synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.), and P rpsU (sequence as shown in SEQ ID NO: 5), respectively, thereby constructing four plasmids which were introduced into the non-resistance gene carrying E. coli competent cells prepared in Example 1.

[0049] After sequencing, single colonies of the four strains were inoculated into LB medium. After culturing at 37°C and 200 rpm for 16-24 hours, they were added to LB medium at an inoculation ratio of 2%-5%. After culturing at 37°C and 200 rpm, the OD 600After the OD600 value was 0.4-0.6, the bacteria were inoculated into LB medium containing different concentrations of apramycin. After being cultured at 37°C and 200 rpm for 16-24 hours, the fluorescence intensity (Ex=488 nm, Em=525 nm) was determined and plotted into a graph. In the fluorescence change trend graph of Figure 1 , Figure 1 The curve of D is the most suitable for the target requirement, that is, under the regulation of the p15A ori medium copy replicon, the fluorescence response effect of the biosensor is the most ideal. cspA Under the regulation of the P cspA (Sequence as shown in SEQ ID NO: 3) is the optimal promoter screened out.

[0050] Example 2.2: Construction and optimization of replicon

[0051] Then, P cspA was selected as the promoter, and the replicon was further screened. Three replicons with different copy numbers were used, that is, RSF ori high copy replicon (sequence as shown in SEQ ID NO: 6, synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.), p15A ori medium copy replicon (sequence as shown in SEQ ID NO: 7, synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.), and pSC101 ori low copy replicon (sequence as shown in SEQ ID NO: 8, synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.). The three constructed plasmids were introduced into the fluorescence E. coli competent cells without resistance genes prepared in Example 1.

[0052] After sequencing, single colonies of the three strains were inoculated into LB medium. After being cultured at 37°C and 200 rpm for 16-24 hours, they were added to LB medium at an inoculation ratio of 2%-5%. After being cultured at 37°C and 200 rpm, the OD600 value was 0.4-0.6, and the fluorescence intensity (Ex=488 nm, Em=525 nm) was determined and plotted into a graph. In the fluorescence change trend graph of 600 , Figure 2 Among the three fluorescence change trend curves of

[0053] Therefore, on the basis of determining the optimal promoter and the optimal replicon in Example 2, an expression cassette composed of an apramycin resistance gene, a promoter, and a replicon was constructed, and the expression cassette was introduced into the fluorescence E. coli competent cells without resistance genes, thereby constructing the apramycin biosensor of the present application.

[0054] Example 3: Dynamic response range determination

[0055] The amikacin biosensor determined in Example 2 was re-validated. Specifically, the amikacin biosensor was put into different concentrations of amikacin LB medium, and the fluorescence intensity (Ex = 488 nm, Em = 525 nm) was determined after incubation at 37°C and 200 rpm for 16-24 hours and plotted into a graph.

[0056] As shown in Figure 3 , the graph trend of Example 3 is basically consistent with that in Example 2, that is, the fluorescence signal gradually decreases with the increase of amikacin concentration.

[0057] Example 4: Specificity verification

[0058] The amikacin biosensor was cultured according to the culture method described in Example 2, and was inoculated into LB medium containing five commonly used antibiotics with different concentrations (amikacin LB medium, spectinomycin LB medium, gentamicin LB medium, ampicillin LB medium, penicillin LB medium). The fluorescence intensity (Ex = 488 nm, Em = 525 nm) was determined after incubation at 37°C and 200 rpm for 16-24 hours, and the corresponding column chart was plotted as shown in Figure 4 . As shown in Figure 4 , it can be seen that the biosensor of the present application can show a good trend of gradually decreasing fluorescence signal with the increase of concentration of amikacin (blue column), and does not show such trend for the other four antibiotics. Therefore, the biosensor of the present application has good specificity for amikacin concentration.

[0059] Example 5: Droplet system application

[0060] In order to study whether the amikacin biosensor of the present application can be applied to the droplet system to further verify the feasibility of the biosensor, the following experiment was carried out.

[0061] The seed liquid of the amikacin biosensor of the present application was cultured according to the culture method described in Example 2, and the OD 600 was adjusted to 0.4-0.6. Then the seed liquid was mixed with 2xLB medium containing different concentrations (0 mg / mL, 1 mg / mL, 5 mg / mL) at a volume ratio of 1:1. Then three concentrations of droplets were generated by high-throughput picoliter droplet single cell sorting system (DREM cell of Luoyang Huqing Tianmu Biotechnology Co., Ltd.). Incubate at 37°C for 3-6 hours. After microscopic examination and sorting by DREM cell.

[0062] As shown in Figure 5As shown, in the microscope examination, the growth of the strain and the change of the fluorescence signal in the droplet can be clearly observed in the bright field and dark field view. The higher the concentration of apramycin, the worse the growth of the biosensor, and the weaker the fluorescence signal. Similarly, Figure 6 As shown, with the increase of the concentration of apramycin, the fluorescence signal of the droplet gradually decreases. These all indicate that the growth of the biosensor of the application is different in different concentrations of apramycin.

[0063] According to the results of Figure 5 and Figure 6 It can be seen that the DERM cell sorting system has good distinguishing effect on droplets with different concentrations of apramycin.

[0064] The results of Example 5 also further verify the specific application feasibility of the apramycin biosensor of the application.

[0065] Summary: The application constructs an apramycin-responsive biosensor based on apramycin resistance genes, which has stable response between batches and has broad application prospects in the screening of high-yield apramycin strains. The application accelerates the pace of screening high-yield apramycin strains, and provides a biosensor skeleton for screening other antibiotics, and potentially accelerates the screening of high-yield strains of antibiotics with corresponding resistance genes.

[0066] Although the embodiments of the application are described above in combination with the drawings, the application is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and guiding, but not limiting. Those skilled in the art can make many forms under the guidance of this specification and without departing from the scope protected by the claims of the application, which all belong to the protection of the application.

[0067] SEQUENCE LISTING

[0068]

[0069]

Claims

1. An expression cassette, wherein, The expression cassette comprises an apramycin resistance gene sequence, a promoter sequence and a replicon sequence.

2. The expression cassette of claim 1, wherein the apramycin resistance gene sequence is set forth in SEQ ID NO:

1.

3. The expression cassette of claim 1 or 2, wherein the promoter sequence is selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NO: 2, a nucleotide sequence set forth in SEQ ID NO: 3, a nucleotide sequence set forth in SEQ ID NO: 4, and a nucleotide sequence set forth in SEQ ID NO:

5.

4. The expression cassette of claim 1 or 2, wherein the promoter sequence is set forth in SEQ ID NO:

3.

5. The expression cassette of any one of claims 1 to 4, wherein the replicon sequence is selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NO: 6, a nucleotide sequence set forth in SEQ ID NO: 7, and a nucleotide sequence set forth in SEQ ID NO:

8.

6. The expression cassette of any one of claims 1 to 4, wherein the replicon sequence is set forth in SEQ ID NO:

7.

7. An apramycin biosensor comprising a plasmid vector, wherein the plasmid vector comprises the expression cassette of any one of claims 1 to 6, preferably the apramycin biosensor further comprises a fluorescent gene, and further preferably the apramycin biosensor is Escherichia coli.

8. A method for preparing an apramycin biosensor, comprising the steps of: providing a fluorescent Escherichia coli without resistance; constructing a plasmid vector comprising the expression cassette of any one of claims 1 to 6; introducing the plasmid vector into the fluorescent Escherichia coli without resistance to obtain a fluorescent Escherichia coli competent cell as an apramycin biosensor.

9. Use of an apramycin biosensor for detecting the concentration of apramycin, wherein the apramycin biosensor is the apramycin biosensor of claim 7.

10. Use according to claim 9, characterized in that, placing the apramycin biosensor into a strain producing apramycin, determining the fluorescence intensity generated by the apramycin biosensor, and determining whether the apramycin biosensor meets the requirements based on the fluorescence intensity data.

10. A method for detecting the concentration of apramycin, comprising the steps of: providing a fluorescent Escherichia coli without resistance; constructing a plasmid vector comprising the expression cassette of any one of claims 1 to 6; introducing the plasmid vector into the fluorescent Escherichia coli without resistance to obtain a fluorescent Escherichia coli competent cell as an apramycin biosensor; placing the apramycin biosensor into a strain producing apramycin, determining the fluorescence intensity generated by the apramycin biosensor, and determining whether the apramycin biosensor meets the requirements based on the fluorescence intensity data.

Images