A bacterial fluorescence sensor for histidine detection and preparation method thereof

Abstract

The present invention discloses a bacterial fluorescent sensor for histidine detection and a preparation method thereof. The present invention selects a bacterial strain lacking histidine synthesis function as a host bacterium, utilizes the characteristic that the expression of its histidine operon is enhanced when the histidine concentration is low, and combines a fluorescent protein gene as a reporter gene to construct a bacterial fluorescent sensor with a specific sensitive response to a histidine-deficient environment. The bacterial fluorescent sensor can perform rapid, sensitive, and in-situ detection of histidine concentration, and has high application value in studying the molecular mechanism of diseases related to abnormal histidine concentration, developing and screening related drugs, and detecting changes in intracellular histidine concentration during pathogen infection.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a bacterial fluorescence sensor for rapid in-situ detection of histidine concentration and a preparation method thereof. Background Art

[0002] As an essential amino acid and precursor of histamine, histidine plays an important role in human growth, metal ion transport and neural regulation. Abnormal levels of histidine or histidine-rich proteins are associated with the occurrence of many diseases. For example, excessive levels of histidine or histidine-rich proteins are associated with phenylketonuria, malaria and thrombotic diseases; while low levels of histidine or histidine-rich proteins are associated with rheumatoid arthritis, epilepsy, chronic kidney disease and advanced liver cirrhosis. The concentration level of histidine in biological samples, especially in living cells, is generally low. The development of a biosensor that responds sensitively to low histidine concentration levels can not only be used to detect the concentration of histidine in cells, cell culture environments and serum, revealing the molecular mechanism of histidine deficiency-related diseases, but also help develop and screen related drugs.

[0003] Currently reported methods for measuring histidine concentration include chromatography, mass spectrometry, electrochemical analysis, and capillary electrophoresis. These methods all require sample pretreatment and cannot be used for rapid, in-situ detection of histidine concentration. Chemical probes based on ligand binding to histidine can be used to detect histidine in extracellular solutions, but additional Cu ions need to be added for intracellular histidine detection. 2+ or Zn 2+ Incubation is performed, and only the elevated histidine in the cell can be detected when histidine is added externally, which makes it difficult to be used for in situ determination of low-concentration endogenous histidine in the cell. In addition, some bioluminescent sensors based on protein-specific binding to histidine can be used for fluorescence imaging when the histidine concentration in living cells increases, but they cannot produce a sensitive response to the intracellular environment lacking histidine. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterial fluorescent sensor that has a rapid, sensitive and specific response to low concentration levels of histidine, which can be used for rapid and sensitive detection of histidine concentrations in serum and cell culture environments and in situ detection of histidine concentrations in living cells, so as to make up for the many shortcomings of existing analytical methods.

[0005] Previous studies have shown that the transcription level of the histidine operon in bacteria is negatively regulated by the histidine level. When the bacterial histidine synthesis function is lost, the transcription level of the bacterial histidine operon is extremely sensitive to the histidine concentration (Li, Z. et al., Salmonella Proteomic Profiling during Infection Distinguishes the Intracellular Environment of Host Cells. Msystems. 2019, 4 (2), 1-14.). Based on this study, the present invention proposes the following technical solution:

[0006] A bacterial fluorescent sensor for histidine detection comprises a host bacterium and a reporter plasmid, wherein the host bacterium is a bacterial strain lacking the histidine synthesis function, and its histidine operon has the characteristic of enhanced expression when the histidine concentration is low; the reporter plasmid contains a promoter of the histidine operon and a reporter gene activated by it, and the reporter gene is a fluorescent protein gene; the reporter plasmid is introduced into the host bacterium, and the expression of the reporter gene is promoted in a histidine-deficient environment, and fluorescence is emitted, thereby responding to a low histidine concentration environment.

[0007] Bacterial strains that can be used as host bacteria of the present invention include, but are not limited to, Salmonella typhimurium SL1344 strains lacking histidine synthesis function. Strains whose histidine operon has the property of enhanced expression at low histidine concentrations can be used in the present invention.

[0008] The promoter of the histidine operon on the reporter plasmid can be the promoter of the histidine operon of Salmonella typhimurium, or the promoter of the histidine operon of other bacteria, such as the promoter of the histidine operon of Escherichia coli. In theory, any promoter regulated by the histidine operon of the host bacteria can be applied to the present invention.

[0009] The reporter gene can be selected from various fluorescent protein genes, and the commonly used enhanced green fluorescent protein (EGFP) gene is used.

[0010] There are no special requirements for the promoter of the histidine operon and the plasmid vector of the reporter gene it initiates. It only needs to contain the basic components of the plasmid such as the replication start site, the selective marker gene (such as the antibiotic resistance gene), etc. The optimal response range of reporter plasmids with different copy numbers to the histidine concentration in the environment will be different.

[0011] The present invention also provides a method for preparing the above-mentioned bacterial fluorescence sensor for histidine detection, comprising the following steps:

[0012] 1) A fragment containing a promoter of a histidine operon and a fragment of a reporter gene are obtained by PCR amplification respectively, and the two fragments are connected to obtain an insert fragment containing a reporter gene driven by the promoter of the histidine operon;

[0013] 2) ligating the insert fragment constructed in step 1) into a plasmid vector to obtain a reporter plasmid;

[0014] 3) The reporter plasmid constructed in step 2) is introduced into a strain whose histidine operon has the property of enhanced expression when the histidine concentration is low, thereby obtaining a bacterial fluorescent sensor for histidine detection.

[0015] In the above step 1), in order to facilitate the subsequent connection of the two fragments, a 15-30 bp long front end sequence of the reporter gene is designed on the 3' end primer of the PCR amplification of the promoter of the histidine operon, and a 15-30 bp long terminal sequence of the promoter of the histidine operon is designed on the 5' end primer of the PCR amplification of the reporter gene. In this way, the two amplified fragments have an overlap of 30-60 bp in length, and then the insertion fragment containing both the promoter of the histidine operon and the reporter gene is obtained by extending them to both ends through PCR.

[0016] In the above step 2), preferably, the insert fragment and the linearized vector are connected to form a plasmid by homologous recombination. During the PCR in step 1), homologous sequences are designed on the 5' end primer of the promoter of the amplified histidine operon and the 3' end primer of the amplified reporter gene, respectively. In step 2), the plasmid vector is used as a template to obtain a linearized vector with homologous sequences at both ends through PCR amplification, and then the linearized vector and the insert fragment are connected to form a plasmid using a homologous recombination kit, and introduced into Escherichia coli, and then the monoclonal strain containing the recombinant plasmid is screened to extract the corresponding plasmid.

[0017] In the above step 3), the reporter plasmid is preferably introduced into the host bacterial strain by electroporation.

[0018] In one embodiment of the present invention, the SL1344 strain of Salmonella typhimurium with a histidine synthesis function loss is selected as the host bacteria, and the characteristic of the histidine operon being enhanced when the histidine concentration is low is utilized, and the reporter gene enhanced green fluorescent protein (EGFP) gene is combined to construct a bacterial fluorescent sensor with a specific sensitive response to a histidine-deficient environment. The specific operation is to construct the promoter of the histidine operon of the SL1344 strain of Salmonella typhimurium and the reporter gene EGFP into a plasmid vector by molecular cloning, and then introduce the constructed reporter plasmid into the SL1344 strain. The characteristic of the high expression of the histidine operon in the SL1344 strain under the histidine-deficient environment is utilized, so that the constructed strain promotes the expression of the reporter gene EGFP under the histidine-deficient environment, emits fluorescence, and realizes the response to the low histidine concentration environment. The bacterial fluorescent sensor can detect the histidine concentration quickly, sensitively, and in situ, and has a high application value in studying the molecular mechanism of diseases related to abnormal histidine concentration, developing and screening related drugs, and detecting the changes in the intracellular histidine concentration during the infection of pathogens.

[0019] Compared with the prior art, the bacterial fluorescence sensor constructed by the present invention has the advantages of no sample processing, rapid in situ detection, and particularly sensitive response to low concentration levels of histidine. In addition, a series of strains with sensitive responses to histidine in different concentration ranges can be constructed as needed to meet the detection needs of different targets, which has obvious progress and advantages:

[0020] (1) Rapid and convenient. The present invention does not require cumbersome sample pretreatment steps, can directly respond to the histidine concentration in the cell culture environment or in the cells, has a short detection time, and is suitable for dynamic tracking and high-throughput detection.

[0021] (2) High sensitivity. The present invention is particularly sensitive to changes in histidine in the low concentration range. In the range of 0 to 50 μM, the fluorescence signal can be measured to respond significantly to changes in concentration. The changes in histidine concentration in many cells are just within this range. Therefore, the bacterial fluorescence sensor has a unique advantage in studying changes in histidine concentration in living cells.

[0022] (3) The concentration response range is adjustable and can be continuously tracked and detected. It has high flexibility and adaptability to the study of different types of cells and different time scales. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Schematic diagram of the construction principle of the plasmid in the bacterial fluorescence sensor of the present invention.

[0024] Figure 2 .Response curve of SL1344-pSB3313 strain to changes in histidine concentration in the culture environment.

[0025] Figure 3 .Response curve (a) and linear range (b) of SL1344-pGEX strain to changes in histidine concentration in the culture environment.

[0026] Figure 4 .The selectivity experimental results of SL1344-pSB3313 strain (a) and SL1344-pGEX strain (b) for histidine and its analogs.

[0027] Figure 5 .Fluorescence imaging results after cells were infected with SL1344-pGEX strain, where: (a) shows the expression level of green fluorescent protein in HeLa cells 1 hour after SL1344-pGEX strain infected them; (b) shows the expression level of green fluorescent protein in macrophages 1 hour after SL1344-pGEX strain infected them; (c) shows the expression level of green fluorescent protein in macrophages 1 hour after SL1344-pGEX strain infected them in culture medium supplemented with 1 mM histidine.

[0028] Figure 6 .Fluorescence imaging results after cells were infected with the SL1344-pSB3313 strain, wherein: (a) shows the expression level of green fluorescent protein in HeLa cells 1 hour after the SL1344-pSB3313 strain infected them; (b) shows the expression level of green fluorescent protein in macrophages 1 hour after the SL1344-pSB3313 strain infected them; (c) shows the expression level of green fluorescent protein in macrophages 1 hour after the SL1344-pSB3313 strain infected them in culture medium supplemented with 1 mM histidine. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail by embodiments in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0030] Example 1 <Construction of bacterial fluorescence sensor>

[0031] The process of constructing plasmids is as follows Figure 1As shown, firstly, pSB3313 plasmid and pGEX-6p-1 plasmid (purchased from Beijing Solebow Technology Co., Ltd.) were used as templates to obtain two different linearized vectors by PCR amplification, including the basic components of plasmids such as replication initiation site, resistance gene, and promoter. Then, fragments of the promoter containing the histidine operon (see SEQ ID No: 1 in the sequence table) and the EGFP gene were obtained by PCR amplification. In order to facilitate the subsequent connection of the two fragments, a 20 bp sequence at the front end or end of the other gene was designed on the corresponding PCR primers of the two fragments, so that the two amplified fragments produced an overlap of 40 bp sequence, and then extended to both ends by PCR to obtain a fragment containing both the promoter of the histidine operon and the EGFP gene. Finally, the linearized vector and the inserted fragment were connected into a plasmid using a one-step homologous recombination kit, and introduced into DH5α chemically competent bacteria, and the bacteria were spread on a resistant LB plate to screen out the monoclonal strain containing the successfully recombined plasmid and extract the corresponding plasmid. Finally, the two plasmids were introduced into the SL1344 strain by electroporation, and two bacterial fluorescent sensors that were sensitive to low histidine concentration were constructed. The SL1344 strain with the plasmid constructed by the vector pSB3313 with a lower copy number was named SL1344-pSB3313, and the SL1344 strain with the plasmid constructed by the vector pGEX-6p-1 with a higher copy number was named SL1344-pGEX.

[0032] Example 2 <Response of bacterial fluorescence sensor to changes in histidine concentration in cell culture environment>

[0033] In this example, two bacterial fluorescence sensors, SL1344-pSB3313 and SL1344-pGEX, constructed by the present invention were used to detect M9 solutions containing different concentrations of histidine. The specific steps are as follows:

[0034] 1. The constructed strains SL1344-pSB3313 and SL1344-pGEX were stored in LB culture medium containing 25% glycerol at -80°C. The frozen strains were inoculated into LB culture medium and cultured in an incubator at 37°C with shaking overnight.

[0035] 2. The next day, the bacteria were subcultured into M9 culture medium containing different concentrations of histidine at a ratio of 1:1 and cultured with shaking in an incubator at 37°C for 30 minutes.

[0036] 3. Take 1 mL of M9 subcultured bacteria, centrifuge and resuspend in 1× PBS, then use an ultrasonic cell disruptor for 5 minutes to completely lyse the bacteria.

[0037] 4. Measure the maximum fluorescence emission wavelength (508 nm) of bacterial lysate under 488 nm excitation in F7000 fluorescence spectrometer

[0038] The fluorescence intensity at .

[0039] Test results such as Figure 2 and Figure 3 As shown in the figure, under the conditions of an excitation slit of 7.5 nm, an emission slit of 5.0 nm, and a photomultiplier voltage of 950 V, the response range of the SL1344-pSB3313 strain to changes in the histidine concentration in the culture medium was measured to be 0 to 1000 μM ( Figure 2 ); SL1344-pGEX strain, under the conditions of excitation slit and emission slit of 5.0nm and photomultiplier voltage of 950V, can produce a very sensitive response to histidine concentrations ranging from 0 to 50.00μM ( Figure 3 ), indicating that the SL1344-pGEX strain can perform quantitative measurements in an environment with extreme histidine deficiency, while the SL1344-pSB3313 strain can monitor changes in histidine concentration at relatively high levels.

[0040] Example 3<Study on the Selectivity of Bacterial Fluorescence Sensor>

[0041] In this example, the selectivity of two bacterial fluorescent sensors SL1344-pSB3313 and SL1344-pGEX constructed by the present invention for histidine detection was studied, and the specific steps are as follows:

[0042] 1. The constructed strains SL1344-pSB3313 and SL1344-pGEX were stored in LB culture medium containing 25% glycerol at -80°C. The frozen strains were inoculated into LB culture medium and cultured in an incubator at 37°C with shaking overnight.

[0043] 2. The next day, the bacteria were subcultured into M9 culture medium containing 50 μM histidine, lysine, arginine and histamine at a ratio of 1:1, and cultured in an incubator at 37°C with shaking for 30 min.

[0044] 3. Take 1 mL of M9 subcultured bacteria, centrifuge and resuspend in 1× PBS, then use an ultrasonic cell disruptor for 5 minutes to completely lyse the bacteria.

[0045] 4. Measure the fluorescence intensity of the bacterial lysate at an excitation wavelength of 488 nm and an emission wavelength of 508 nm in a F7000 fluorescence spectrometer.

[0046] Test results such as Figure 4As shown, when 50 μM histidine was added to the M9 culture medium, the expression level of bacterial green fluorescent protein decreased significantly compared with that without adding histidine, while when 50 μM of the other two positively charged amino acids (lysine and arginine) or histidine analog histamine were added, the expression level of bacterial green fluorescent protein did not change significantly, indicating that the bacterial fluorescent sensor constructed by the present invention has good selectivity for histidine.

[0047] Example 4 <In situ response of bacterial fluorescence sensor to changes in intracellular histidine concentration>

[0048] In this example, two bacterial fluorescent sensors SL1344-pSB3313 and SL1344-pGEX constructed by the present invention were used to perform in situ detection on HeLa cells (with an intracellular histidine concentration of about 200 μM) and macrophage RAW264.7 cells (with an intracellular histidine concentration of about 60 μM), respectively. The specific steps are as follows:

[0049] 1. HeLa cells and RAW264.7 cells were used as hosts respectively. When the cells grew to a density of 70-80%, they could be used for infection experiments.

[0050] 2. Before infection, remove the culture medium in the culture dish, add 37℃ preheated HBSS, shake gently and wash twice, and incubate in 37℃ preheated HBSS for 30 minutes.

[0051] 3. Cultivate 1 mL to OD 600 The strain with a molar mass of 0.9 was centrifuged at 3000×g for 3 min, resuspended in 1 mL of HBSS preheated at 37°C, added to the cell culture dish, and incubated at 37°C for 30 min for infection. The ratio of bacteria to cells (MOI) during the infection process was approximately 100:1.

[0052] 4. After infection, wash gently once with HBSS preheated at 37°C, then add DMEM preheated at 37°C containing 100 μg / mL gentamicin and incubate for 1 hour to remove extracellular bacteria. The time point of adding gentamicin is defined as the infection start time point.

[0053] 5. After 1 h, the culture medium was replaced with DMEM containing 20 μg / mL gentamicin preheated at 37°C and cultured at 37°C for 30 min.

[0054] 6. Before observing with a confocal microscope, stain the cell nuclei with a nuclear dye. Add 100× dye to the culture medium for 10 minutes, then rinse the cells with the culture medium and observe the fluorescence of intracellular bacteria under a microscope at 488nm excitation / 508nm emission.

[0055] Figure 5 and Figure 6 The fluorescence imaging results of the SL1344-pGEX strain and the SL1344-pSB3313 strain are shown respectively.

[0056] Depend on Figure 5 and Figure 6 It can be seen that the intensity of green fluorescence emitted by bacteria in macrophages is significantly higher than that in HeLa cells. This is consistent with the literature report that the concentration of histidine in HeLa cells is about 200μM and the concentration of histidine in macrophage RAW264.7 cells is about 60μM. To further confirm, 1mM histidine was supplemented in the culture environment of macrophages, and the intensity of green fluorescence emitted by bacteria was significantly weakened, indicating that the lower histidine concentration of macrophages itself led to the higher expression of green fluorescent protein in the bacterial fluorescent sensor. When the histidine concentration increased, the expression of green fluorescent protein decreased. SEQUENCE LISTING <110> Beijing University <120> A bacterial fluorescence sensor for histidine detection and preparation method thereof <130> WX2021-03-158 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 269 <212> DNA <213> Salmonella typhimurium <400> 1 gcgcttgctt taaggcgtaa aagtggttta ggttaaaagg tatcaaatga ataagcattc 60 atcggaattt ttatgacacg cgttcaattt aaacaccacc atcatcacca tcatcctgac 120 tagtctttca ggcgatgtgt gctggaagac attcagatct tccagtggtg catgaacgca 180 tgagaaagcc cccggaagat catcttccgg gggctttttt tttggcgcgc gatacagcc 240 ggttcagaca ggataaagag gaacgcaga 269

Claims

1. A method for preparing a bacterial fluorescent sensor for histidine detection, wherein the bacterial fluorescent sensor for histidine detection comprises a host bacterium and a reporter plasmid, wherein: The host bacteria is a bacterial strain lacking the histidine synthesis function, and its histidine operon has the characteristic of enhanced expression when the histidine concentration is low; the reporter plasmid contains the promoter of the histidine operon and the reporter gene it activates, and the reporter gene is a fluorescent protein gene; the reporter plasmid is introduced into the host bacteria, and the expression of the reporter gene is promoted in an environment lacking histidine, and fluorescence is emitted, thereby responding to the low histidine concentration environment; the preparation method thereof comprises the following steps: 1) A fragment containing a promoter of a histidine operon and a fragment of a reporter gene are obtained by PCR amplification respectively, and the two fragments are connected to obtain an insert fragment containing a reporter gene driven by the promoter of the histidine operon; specifically, a 15-30 bp front end sequence of the reporter gene is designed on the 3' end primer of the PCR amplification of the promoter of the histidine operon, and a 15-30 bp terminal sequence of the promoter of the histidine operon is designed on the 5' end primer of the PCR amplification of the reporter gene, the two amplified fragments produce a 30-60 bp overlap sequence, and then the insert fragment is extended to both ends by PCR to obtain the insert fragment; 2) Connect the insert fragment constructed in step 1) into the plasmid vector to obtain a reporter plasmid; 3) Introducing the reporter plasmid constructed in step 2) into the host bacteria to obtain a bacterial fluorescent sensor for histidine detection.

2. The preparation method according to claim 1, characterized in that The host bacteria is the SL1344 strain of Salmonella typhimurium.

3. The preparation method according to claim 1, characterized in that: The promoter of the histidine operon is a promoter of the histidine operon of Salmonella typhimurium or a promoter of the histidine operon of Escherichia coli.

4. The preparation method according to claim 1, characterized in that: The sequence of the promoter of the histidine operon is shown as SEQ ID No: 1 in the sequence listing.

5. The preparation method according to claim 1, characterized in that: The reporter gene is an enhanced green fluorescent protein gene.

6. The preparation method according to claim 1, characterized in that: In step 2), the insert fragment and the linearized vector are connected into a plasmid using a homologous recombination method to obtain a reporter plasmid.

7. The preparation method according to claim 6, characterized in that: Step 1) homologous sequences are designed on the 5' end primer of the promoter of the histidine operon and the 3' end primer of the reporter gene for PCR amplification, respectively, to obtain an insert fragment with homologous sequences at both ends; Step 2) using the plasmid vector as a template, a linearized vector with homologous sequences at both ends is obtained by PCR amplification; the linearized vector and the insert fragment are connected into a plasmid using a homologous recombination kit, and introduced into Escherichia coli, and then a monoclonal strain containing the recombinant plasmid is screened to extract the corresponding plasmid.

8. The preparation method according to claim 1, characterized in that: Step 3) electroporation is used to introduce the reporter plasmid into the host bacteria.

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