Patent Publication Number: US-2023159980-A1

Title: Method for detecting l-serine based on cysteine desulfurase-containing living escherichia coli cell

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This patent application claims the benefit and priority of Chinese Patent Application No. 202111282167.8, filed on Nov. 1, 2021, the disclosure of which is incorporated by reference herein in its entirety as part of the present application. 
     TECHNICAL FIELD 
     The present disclosure relates to the technical field of amino acid detection, in particular to a method for detecting L-serine based on cysteine desulfurase-containing living  Escherichia coli  cells. 
     BACKGROUND ART 
     L-serine is a kind of polar amino acid, which belongs to a non-essential polar amino acid in the human body. It participates in the biosynthesis of human proteins, purines, pyrimidines and phospholipids, and plays an important role in immune regulation, tumor metabolism and other processes. It has been indicated that the content of L-serine in urine is expected to become a tumor marker for some cancers, which can be used to assist in diagnosing tumors or judging prognosis. In addition, L-serine is also widely used in amino acid infusions, nutritional additives and deluxe cosmetics. Therefore, providing a quick and easy detection method for determining the content of L-serine in mixed amino acids, especially a detection technology capable of eliminating the interference of D-serine, cycloserine, serine analogs and serine derivatives, is extremely important for the development and utilization of L-serine. 
     At present, methods for determining amino acid content inside and outside of China are mainly divided into instrumental methods, chromogenic methods, chemiluminescence methods, and enzymatic methods. Instrumental methods usually include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and automatic amino acid analyzer. The chromogenic methods include chromotropic acid-spectrophotometry, paper chromatography-spectrophotometry, and ninhydrin method. Chemiluminescence method is mainly capillary electrophoresis coupling with electrochemiluminescence. Enzymatic methods include serine aminotransferase method and cystathionine lyase method. 
     Among them, instrumental methods such as liquid chromatography, gas chromatography, and chromatography-mass spectrometry have good sensitivity and high accuracy, but require expensive equipment, specialized laboratories, professionally trained laboratory technicians, high maintenance costs, and relatively high laboratory consumables. As the most basic and traditional detection method, ninhydrin method is easy to operate and quick to react, but it has high requirements on reaction conditions, and requires precise control of reaction temperature, pH, and time. Moreover, the method has different sensitivities to different types of amino acids and is not suitable for analysis of samples that require high precision. Fluorescence quenching method can avoid the interference of most amino acids, but the sample needs to undergo complex phosphorylation pretreatment, and the detection result has a large error. The paper chromatography-spectrophotometry is easy to operate, but it is not suitable for the analysis and detection of large quantities of samples due to its poor stability. Chromotropic acid-spectrophotometry has fast reaction speed, simple operation, and high accuracy, but poor anti-interference ability. 
     Enzymatic methods have excellent specificity and accuracy, but the currently used enzymatic methods have many reaction steps and many factors that affect the determination, and the detection signal often cannot be directly observed or directly measured. In addition, enzymes have poor stability and are not easy to store. Therefore, it is very necessary to develop a living cell enzymatic method for determining L-serine which is simple and can be directly observed with the naked eye after the reaction. 
     INCORPORATION BY REFERENCE 
     Submitted with the present application is an electronically filed sequence listing via the Patent Center as an CML formatted sequence listing, entitled “GWP20220400808.xml”, created Sep. 29, 2022, and 3,182 bytes in size. The sequence listing is part of the specification filed herewith and is incorporated by reference in its entirety. 
     SUMMARY 
     An objective of the present disclosure is to provide a method for detecting L-serine based on cysteine desulfurase-containing living  E. coli  cells. By incubating the cysteine desulfurase-containing living  E. coli  cells with L-serine, a new living cell detection method of L-serine is developed to realize qualitative or semi-quantitative detection with naked eyes, as well as quantitative detection of L-serine content. 
     To achieve the above objective, the present disclosure provides the following solution: 
     The present disclosure provides a method for detecting L-serine based on cysteine desulfurase-containing living  E. coli  cells, including steps of: incubating an unknown sample with the cysteine desulfurase-containing living  E. coli  cells to produce a red substance, and qualitatively or semi-quantitatively detecting L-serine content in the unknown sample according to color changes of the red substance of the living  E. coli  cells, or quantitatively detecting L-serine content in the unknown sample by measuring absorbance of a lysate of the living  E. coli  cells. 
     Preferably, the unknown sample and the cysteine desulfurase-containing living  E. coli  cells may be mixed and incubated in a volume ratio of 1:(5-10). 
     Preferably, incubation conditions may be as follows: cells may be cultured at 32-37° C. and 200-250 rpm under shaking for 2-12 h. 
     Preferably, incubation of the unknown sample with the cysteine desulfurase-containing living  E. coli  cells to produce the red substance may be followed by directly observing whether a red color is generated with the naked eye and qualitatively determining whether the unknown sample contains L-serine, and comparing the depth of a generated red color with a colorimetric card constructed with an L-serine standard solution to semi-quantitatively determine a range of the L-serine content in the unknown sample. 
     Preferably, after the unknown sample is incubated with the cysteine desulfurase-containing living  E. coli  cells to produce the red substance, a supernatant is collected by sonication and centrifugation, absorbance of the supernatant is measured, and the absorbance is substituted into a standard curve constructed by the L-serine standard solution to quantitatively determine the L-serine content in the unknown sample. 
     Preferably, the cysteine desulfurase-containing living  E. coli  cells may be prepared by the following steps: 
     transforming a pBAD expression vector pBISCS containing cysteine desulfurase IscS into  E. coli  MC4100 to construct a pBISCS/MC4100 strain; 
     inoculating the pBISCS/MC4100 strain into LB broth supplemented with ampicillin to obtain a bacterial suspension with an OD600 of 0.6-0.8, and inducing the bacterial suspension with L-arabinose for 2-3 h to harvest cells; and 
     washing the cells once or twice with M9 Buffer, and resuspending the cells in M9 Buffer supplemented with chloramphenicol and ampicillin to obtain the cysteine desulfurase-containing living  E. coli  cells. 
     Preferably, a gene sequence encoding the cysteine desulfurase IscS is shown in SEQ ID NO: 1. 
     The present disclosure provides the following technical effects: 
     According to the inventors&#39; previous research, it is found that  E. coli  cysteine desulfurase is expressed in an IscA/SufA double-deficient bacterium. The enzyme turns red when observed with the naked eye, and there is a stable characteristic absorption peak at 528 nm when scanned by the UV-Vis spectrophotometer, but the mechanism of intracellular production of the red substance is not clear. Through further in-depth research, it is found that a main factor for the production of this stable red substance in cells is L-serine, and more importantly, the depth of redness and the height of the absorption peak at 528 nm show a dose-dependent relationship with the L-serine added in the medium. Based on this original discovery, the inventors optimize an L-serine enzymatic detection technology, and develop a method for detecting L-serine based on cysteine desulfurase-containing living  E. coli  cells in the present application. 
     Specifically, in the present disclosure, an L-serine sample is incubated with the cysteine desulfurase-containing living  E. coli  cells to produce a red substance, and the L-serine in the sample is qualitatively and quantitatively determined by the color depth of the red substance or by measuring the absorbance of a bacterial cell lysate at 528 nm. The process designed by the present disclosure is simple, with few reaction steps and stable enzyme activity in living cells. Not only can the process achieve intuitive qualitative detection and precise quantification, but also can effectively prevent the interference of D-serine, cycloserine, serine analogs and other amino acids. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       In order to illustrate the examples of the present disclosure or the technical solution in the prior art more clearly, the accompanying drawings required in the examples will be briefly introduced below. Obviously, the drawings in the following description are only some of the present disclosure. Other drawings can also be obtained by those of ordinary skill in the art without creative work based on these drawings. 
         FIG.  1    illustrates the results of reactions of different amino acids with whole cells, where IscS is a control without amino acid addition, the capital letters after “+” are the abbreviations of amino acid types, and configurations thereof are all L-configurations; 
         FIGS.  2 A and  2 B  illustrate the determination of the optimal incubation time and linear range; panel A is the determination of the optimal incubation time, where excess L-serine (4 mM) is added to react for different time to take out for absorbance measurement at 528 nm and plotting of a curve with time; panel B is determination of the optimal linear range, where different concentrations of amino acids react with living cells for 3 h, and the absorbance is measured at 528 nm to plot a concentration-dependent curve. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     The technical solution of the present disclosure will now be specifically described by way of examples. However, they should not be construed as limiting the present disclosure, but should be understood as more detailed descriptions of certain aspects, characteristics and embodiments of the present disclosure. 
     The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified. 
     Example 1 Method for Quantitatively Detecting L-Serine Based on Cysteine Desulfurase-Containing Living  E. coli  Cells 
     1. Reactive Live Cell Preparation: 
     A pBAD expression vector pBISCS containing cysteine desulfurase IscS (the gene sequence encoding IscS is shown in SEQ ID NO: 1) was transformed into  E. coli  MC4100, namely a pBISCS/MC4100 strain, designated WMU-013. 
     The preserved  E. coli  WMU-013 was inoculated into LB broth containing 100 μg/mL ampicillin, and cultured at 37° C. and 250 rpm for 12-16 h overnight under shaking, and the bacterial suspension cultured overnight was diluted 1:100 to 500 mL of freshly prepared LB broth containing 100 μg/mL ampicillin; under the same conditions, the system was continued to culture until OD600 nm was 0.6; after being induced with 0.02% L-arabinose for 3 h, the cells were collected by centrifugation, washed with 50 mL of M9 Buffer (12.8 g/L Na 2 HPO 4 ·7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, and 1 g/L NH 4 Cl) once, resuspended in the same buffer (250 mL) supplemented with 34 mg/mL chloramphenicol and 100 mg/mL ampicillin, and aliquoted in 50 mL/part for later use. 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO: 1: 
               
               
                   
                 ATGGAATTACCGATTTATCTCGACTACTCCGCAAC 
               
               
                   
                   
               
               
                   
                 CACGCCGGTGGACCCGCGTGTTGCCGAGAAAATGA 
               
               
                   
                   
               
               
                   
                 TGCAGTTTATGACGATGGACGGAACCTTTGGTAAC 
               
               
                   
                   
               
               
                   
                 CCGGCCTCCCGTTCTCACCGTTTCGGCTGGCAGGC 
               
               
                   
                   
               
               
                   
                 TGAAGAAGCGGTAGATATCGCCCGTAATCAGATTG 
               
               
                   
                   
               
               
                   
                 CCGATCTGGTCGGCGCTGATCCGCGTGAAATCGTC 
               
               
                   
                   
               
               
                   
                 TTTACCTCTGGTGCAACCGAATCTGACAACCTGGC 
               
               
                   
                   
               
               
                   
                 GATCAAAGGTGCAGCCAACTTTTATCAGAAAAAAG 
               
               
                   
                   
               
               
                   
                 GCAAGCACATCATCACCAGCAAAACCGAACACAAA 
               
               
                   
                   
               
               
                   
                 GCGGTACTGGATACCTGCCGTCAGCTGGAGCGCGA 
               
               
                   
                   
               
               
                   
                 AGGTTTTGAAGTCACCTACCTGGCACCGCAGCGTA 
               
               
                   
                   
               
               
                   
                 ACGGCATTATCGACCTGAAAGAACTTGAAGCAGCG 
               
               
                   
                   
               
               
                   
                 ATGCGTGACGACACCATCCTCGTGTCCATCATGCA 
               
               
                   
                   
               
               
                   
                 CGTAAATAACGAAATCGGCGTGGTGCAGGATATCG 
               
               
                   
                   
               
               
                   
                 CGGCTATCGGCGAAATGTGCCGTGCTCGTGGCATT 
               
               
                   
                   
               
               
                   
                 ATCTATCACGTTGATGCAACCCAGAGCGTGGGTAA 
               
               
                   
                   
               
               
                   
                 ACTGCCTATCGACCTGAGCCAGTTGAAAGTTGACC 
               
               
                   
                   
               
               
                   
                 TGATGTCTTTCTCCGGTCACAAAATCTATGGCCCG 
               
               
                   
                   
               
               
                   
                 AAAGGTATCGGTGCGCTGTATGTACGTCGTAAATC 
               
               
                   
                   
               
               
                   
                 GCGCGTACGCATCGAAGCGCAAATGCACGGCGGCG 
               
               
                   
                   
               
               
                   
                 GTCACGAGCGCGGTATGCGTTCCGGCACTCTGCCT 
               
               
                   
                   
               
               
                   
                 GTTCACCAGATCGTCGGAATGGGCGAGGCCTATCG 
               
               
                   
                   
               
               
                   
                 CATCGCAAAAGAAGAGATGGCGACCGAGATGGAAC 
               
               
                   
                   
               
               
                   
                 GTCTGCGCGGCCTGCGTAACCGTCTGTGGAACGGC 
               
               
                   
                   
               
               
                   
                 ATCAAAGATATCGAAGAAGTTTACCTGAACGGTGA 
               
               
                   
                   
               
               
                   
                 CCTGGAACACGGTGCGCCGAACATTCTCAACGTCA 
               
               
                   
                   
               
               
                   
                 GCTTCAACTACGTTGAAGGTGAGTCGCTGATTATG 
               
               
                   
                   
               
               
                   
                 GCGCTGAAAGACCTCGCAGTTTCTTCAGGTTCCGC 
               
               
                   
                   
               
               
                   
                 CTGTACGTCAGCAAGCCTCGAACCGTCCTACGTGC 
               
               
                   
                   
               
               
                   
                 TGCGCGCGCTGGGGCTGAACGACGAGCTGGCACAT 
               
               
                   
                   
               
               
                   
                 AGCTCTATCCGTTTCTCTTTAGGTCGTTTTACTAC 
               
               
                   
                   
               
               
                   
                 TGAAGAAGAGATCGACTACACCATCGAGTTAGTTC 
               
               
                   
                   
               
               
                   
                 GTAAATCCATCGGTCGTCTGCGTGACCTTTCTCCG 
               
               
                   
                   
               
               
                   
                 CTGTGGGAAATGTACAAGCAGGGCGTGGATCTGAA 
               
               
                   
                   
               
               
                   
                 CAGCATCGAATGGGCTCATCATCATCATCATCATT 
               
               
                   
                   
               
               
                   
                 GA 
               
            
           
         
       
     
     2. Preparation of 1 M L-Serine Standard 
     3. Determination of Optimal Incubation Time and Linear Range 
     A. Determination of Incubation Time 
     Unknown L-serine (4 mM) was mixed with the living cell suspension in a ratio of 1:10, and cultured at 37° C. and 250 rpm for different times under shaking; the cells were sonicated and centrifuged to take the supernatant, and the absorbance of the supernatant at 528 nm was measured, and a curve was plotted with time as the horizontal axis and the absorbance value at 528 nm as the vertical axis. 
     B. Determination of Linear Range 
     The L-serine standard solutions of a series of concentrations were mixed with the living cell suspension, and incubated at 37° C. and 250 rpm for 3 h under shaking; the color depth was observed with the naked eye and photographed to establish a colorimetric card; the cells were sonicated and centrifuged to take the supernatant, and the absorbance of the supernatant at 528 nm was measured, and a concentration-dependent curve was plotted with concentration as the horizontal axis and the absorbance value at 528 nm as the vertical axis. 
     4. Plotting of a Standard Curve 
     A standard curve was plotted with the concentration of the standard solution as the abscissa and the absorbance at 528 nm as the ordinate, and curve fitting was conducted to obtain a curve equation and an R 2  value. 
     5. Quantification of the Concentration of the Unknown Sample 
     The L-serine concentration in the unknown sample was obtained according to the equation and the absorbance value of the unknown sample tube. 
     6. Results and Analysis 
     The naked eye observation and the results of the colorimetric card showed that after the standard L-serine in the concentration range of 0-1 mM reacted with the living cell suspension, the color turned from pale yellow to pale pink and gradually darkened to red. By observing with the naked eye, the color change had good discrimination, and different colors and their depths could reflect the presence or absence of L-serine well, as well as the level of concentration. It can be seen that the experimental method for qualitative/semi-quantitative detection of L-serine of the present disclosure is feasible, convenient and efficient. 
     As shown in  FIG.  2 A , L-serine responds quickly after incubation with the living cell suspension, shows a linear increase within 0-3 h, then plateaus stably, and decreases after 12 h. In order to reduce the measurement time, 3 h was selected as the optimal incubation time. 
     As shown in  FIG.  2 B , different concentrations of L-serine are incubated with living cell suspensions for 3 h, and the absorbance at 528 nm representing a red color in the supernatant gradually increases with the increase of L-serine concentration, and increases linearly in the concentration range of 0-1 mM, and then plateaus. Thus, it can be seen that the linear range of L-serine is 0-1 mM. 
     Example 2 Detection of the Ability to Resist the Interference of Other Amino Acids 
     500 μL each of unknown L-serine and other L-amino acids (4 mM) were mixed with 500 μL of living cell suspensions, respectively, and incubated on a shaker at 37° C. and 250 rpm for 3 h, and the color depth was observed with the naked eye and photographed. 
     As shown in  FIG.  1   , only L-serine appears red only after incubation with the living cell suspension, and other amino acids and the control without amino acids all appear pale yellow, indicating that this method can directly observe the generation of red with the naked eye to determine whether there is L-serine or not. At the same time, it is not difficult to analyze that this method has a good ability to resist the interference of other amino acids and has good specificity. In addition, other types of amino acids were also detected by this method. Experimental methods demonstrated that the method had an excellent ability to resist the interference of D-serine, cycloserine and serine derivatives. The above results show that the method is feasible to qualitatively detect L-serine in a simple and easy manner, and has excellent specificity. 
     The above examples are only intended to describe the preferred implementations of the present disclosure, but not to limit the scope of the present disclosure. Various alterations and improvements made by those of ordinary skill in the art based on the technical solution of the present disclosure without departing from the design spirit of the present disclosure shall fall within the scope of the appended claims of the present disclosure. 
     
       
         
           
               
               
            
               
                 Sequence Listing Information: 
                   
               
               
                  DTD Version: V1_3 
               
               
                  File Name: GWP20220400808.xml 
               
               
                  Software Name: WIPO Sequence 
               
               
                  Software Version: 2.1.2 
               
               
                  Production Date: 2022 Sep. 29 
               
               
                 General Information: 
               
               
                  Current application/Applicant file reference: GWP20220400808 
               
               
                  Earliest priority application/IP Office: CN 
               
               
                  Earliest priority application/Application number: 202111282167.8 
               
               
                  Earliest priority application/Filing date: 2021-11-01 
               
               
                  Applicant name: Wenzhou Medical College 
               
               
                  Applicant name/Language: en 
               
               
                 Invention title: METHOD FOR DETECTING L-SERINE BASED ON CYSTEINE 
               
               
                 DESULFURASE-CONTAINING LIVING  ESCHERICHIA COLI  CELL (en) 
               
               
                  Sequence Total Quantity: 1 
               
               
                 Sequences: 
               
               
                  Sequence Number (ID): 1 
               
               
                  Length: 1227 
               
               
                  Molecule Type: DNA 
               
               
                  Features Location/Qualifiers: 
               
               
                   -source, 1..1227 
               
               
                    &gt; mol_type, other DNA 
               
               
                    &gt; note, Gene sequence encoding IscS 
               
               
                    &gt; organism, synthetic construct 
               
               
                 Residues: 
               
               
                 atggaattac cgatttatct cgactactcc gcaaccacgc cggtggaccc gcgtgttgcc   60 
               
               
                   
               
               
                 gagaaaatga tgcagtttat gacgatggac ggaacctttg gtaacccggc ctcccgttct  120 
               
               
                   
               
               
                 caccgtttcg gctggcaggc tgaagaagcg gtagatatcg cccgtaatca gattgccgat  180 
               
               
                   
               
               
                 ctggtcggcg ctgatccgcg tgaaatcgtc tttacctctg gtgcaaccga atctgacaac  240 
               
               
                   
               
               
                 ctggcgatca aaggtgcagc caacttttat cagaaaaaag gcaagcacat catcaccagc  300 
               
               
                   
               
               
                 aaaaccgaac acaaagcggt actggatacc tgccgtcagc tggagcgcga aggttttgaa  360 
               
               
                   
               
               
                 gtcacctacc tggcaccgca gcgtaacggc attatcgacc tgaaagaact tgaagcagcg  420 
               
               
                   
               
               
                 atgcgtgacg acaccatcct cgtgtccatc atgcacgtaa ataacgaaat cggcgtggtg  480 
               
               
                   
               
               
                 caggatatcg cggctatcgg cgaaatgtgc cgtgctcgtg gcattatcta tcacgttgat  540 
               
               
                   
               
               
                 gcaacccaga gcgtgggtaa actgcctatc gacctgagcc agttgaaagt tgacctgatg  600 
               
               
                   
               
               
                 tctttctccg gtcacaaaat ctatggcccg aaaggtatcg gtgcgctgta tgtacgtcgt  660 
               
               
                   
               
               
                 aaatcgcgcg tacgcatcga agcgcaaatg cacggcggcg gtcacgagcg cggtatgcgt  720 
               
               
                   
               
               
                 tccggcactc tgcctgttca ccagatcgtc ggaatgggcg aggcctatcg catcgcaaaa  780 
               
               
                   
               
               
                 gaagagatgg cgaccgagat ggaacgtctg cgcggcctgc gtaaccgtct gtggaacggc  840 
               
               
                   
               
               
                 atcaaagata tcgaagaagt ttacctgaac ggtgacctgg aacacggtgc gccgaacatt  900 
               
               
                   
               
               
                 ctcaacgtca gcttcaacta cgttgaaggt gagtcgctga ttatggcgct gaaagacctc  960 
               
               
                   
               
               
                 gcagtttctt caggttccgc ctgtacgtca gcaagcctcg aaccgtccta cgtgctgcgc 1020 
               
               
                   
               
               
                 gcgctggggc tgaacgacga gctggcacat agctctatcc gtttctcttt aggtcgtttt 1080 
               
               
                   
               
               
                 actactgaag aagagatcga ctacaccatc gagttagttc gtaaatccat cggtcgtctg 1140 
               
               
                   
               
               
                 cgtgaccttt ctccgctgtg ggaaatgtac aagcagggcg tggatctgaa cagcatcgaa 1200 
               
               
                   
               
               
                 tgggctcatc atcatcatca tcattga                                     1227 
               
               
                 END