Patent Publication Number: US-2023159940-A1

Title: Culture medium for inducing increase in plasmid copy number and use thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to Chinese Patent Application No. 201811610822.6, entitled “CULTURE MEDIUM FOR INDUCING INCREASE OF PLASMID COPY NUMBER AND USE THEREOF” filed with the China National Intellectual Property Administration on Dec. 27, 2018, which is incorporated herein by reference in its entirety. 
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Dec. 13, 2021, is named “Sequence Listing-20793-0012US00” and is 11,023 bytes in size. 
     BACKGROUND 
     Technical Field 
     The present invention relates to the field of biotechnologies, and specifically, to a culture medium for inducing an increase in a plasmid copy number and use thereof. 
     Related Art 
     In the field of gene synthesis, about 10% or more of gene sequences produce toxicities to host  Escherichia coli . These toxicities are usually introduced by heterologous genes expressed into proteins by the host to affect the physiological metabolism of the host and further the growth of the host. To resist toxic genes, the host  Escherichia coli  tends to retain plasmids with gene mutation or gene deletion, resulting in reduced stability of the genes and plasmids. The use of low-copy plasmids in production can effectively reduce expression of the toxic genes, and improve growth viability of the host and stability of genes. However, the low-copy plasmids have a low yield and are difficult to extract, thereby affecting production efficiency. To resolve the problem, it is necessary to rapidly induce the low-copy plasmids to have a high copy number before plasmid extraction, thereby improving the plasmid yield. However, existing methods for inducing an increase in a plasmid copy number require a plurality of operations such as transfer, OD600 quantification, and addition of an inducer, which are relatively complex and have difficulty in meeting requirements of actual high-throughput production. 
     SUMMARY 
     To resolve the problem of cumbersome operations of an existing plasmid copy number induction system, the present invention provides a culture medium for inducing an increase in a plasmid copy number and use thereof, which improves production efficiency while simplifying operations. 
     An aspect of the present invention provides a culture medium for inducing an increase in a plasmid copy number, including tryptone, a yeast extract, glucose, sodium chloride, and arabinose. 
     In some implementations, the glucose has a concentration of from 0.1 to 10 g/L, preferably from 1 to 5 g/L, and more preferably from 0.5 to 2 g/L. 
     In some implementations, the glucose has a concentration of from 0.1 to 10 g/L. In some implementations, the glucose has a concentration of from 1 to 5 g/L. In some implementations, the glucose has a concentration of from 0.5 to 2 g/L. In some implementations, the glucose has a concentration of 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6 g/L, 1.7 g/L, 1.8 g/L, 1.9 g/L, or 2 g/L. In some implementations, the glucose has a concentration of 0.5 g/L. In some implementations, the glucose has a concentration of 1 g/L. In some implementations, the glucose has a concentration of 2 g/L. In some implementations, the glucose has a concentration of 10 g/L. 
     In some other implementations, the arabinose has a concentration of from 0.1 to 10 g/L, preferably from 1 to 5 g/L, and more preferably from 0.3 to 5 g/L. 
     In some other implementations, the arabinose has a concentration of from 0.1 to 10 g/L. In some implementations, the arabinose has a concentration of from 1 to 5 g/L. In some implementations, the arabinose has a concentration of from 0.3 to 0.75 g/L. In some implementations, the arabinose has a concentration of 0.3 g/L, 0.35 g/L, 0.4 g/L, 0.45 g/L, 0.5 g/L, 0.55 g/L, 0.6 g/L, 0.65 g/L, 0.7 g/L, or 0.75 g/L. In some implementations, the arabinose has a concentration of 0.3 g/L. In some implementations, the arabinose has a concentration of 0.6 g/L. In some implementations, the arabinose has a concentration of 0.75 g/L. In some implementations, the arabinose has a concentration of 1 g/L. In some implementations, the arabinose has a concentration of 2 g/L. In some implementations, the arabinose has a concentration of 5 g/L. 
     In some other implementations, the tryptone has a concentration of from 1 to 15 g/L. 
     In some other preferred implementations, the tryptone has a concentration of from 5 to 10 g/L. 
     In some other implementations, the yeast extract has a concentration of from 1 to 15 g/L. 
     In some other preferred implementations, the yeast extract has a concentration of from 5 to 10 g/L. 
     In some other implementations, the sodium chloride has a concentration of from 1 to 15 g/L. 
     In some other preferred implementations, the sodium chloride has a concentration of from 5 to 10 g/L. 
     In some more preferred implementations, the culture medium further includes one or more of magnesium chloride, potassium chloride, ferric chloride, or calcium chloride. 
     In some implementations, the plasmid is a plasmid including a replication origin oriV. 
     In some implementations, the plasmid is a single-copy plasmid including a replication origin oriV. 
     In some implementations, the plasmid is a single-copy pCCIBAC plasmid. 
     Another aspect of the present invention provides a method for inducing an increase in a plasmid copy number, including the following steps: 
     Step (1): transforming  Escherichia coli  with a plasmid; and 
     Step (2): inoculating the  Escherichia coli  obtained in Step (1) into the culture medium for culturing under conditions suitable for culture. 
     In some implementations, in Step (1), the plasmid is transformed into competent  Escherichia coli.    
     In some other implementations, in Step (2), the  Escherichia coli  is preferably EPI300  Escherichia coli  or EPI400  Escherichia coli.    
     In some other implementations, in Step (2), a culture temperature is from 20 to 37° C. 
     In some other implementations, in Step (2), a culture temperature is from 25 to 37° C. 
     In some other implementations, in Step (2), a culture time is from 4 to 6 h. 
     In some specific implementations, in Step (1), a single-copy plasmid including a replication origin oriV is transformed into an  Escherichia coli  strain, and in Step (2), the  Escherichia coli  is cultured at a temperature of from 20 to 37° C. in a culture medium including glucose with a concentration of from 1 to 5 g/L, arabinose with a concentration of from 1 to 5 g/L, tryptone with a concentration of from 1 to 15 g/L, a yeast extract with a concentration of from 1 to 15 g/L, and sodium chloride with a concentration of from 1 to 15 g/L. 
     In some other specific implementations, in Step (1), the pCCIBAC plasmid is transformed into EPI300  Escherichia coli  or EPI400  Escherichia coli , and in Step (2), the  Escherichia coli  is cultured at a temperature of from 25 to 37° C. in a culture medium including glucose with a concentration of from 0.5 to 2 g/L, arabinose with a concentration of from 0.3 to 0.75 g/L, tryptone with a concentration of from 5 to 10 g/L, a yeast extract with a concentration of from 5 to 10 g/L, and NaCl with a concentration of from 5 to 10 g/L. 
     Still another aspect of the present invention provides use of the culture medium in inducing an increase in a plasmid copy number. 
     The present invention provides a novel culture medium that can induce an increase in a plasmid copy number and improve the plasmid yield. In addition, when the culture medium is used for inducing an increase in a plasmid copy number, operation steps are simple, a culture time is greatly shortened, and a plurality of operations, such as transfer, OD600 quantification, and addition of an inducer, are not required. When the concentration of glucose is lower than 2 g/L, and the concentration of arabinose is lower than 0.75 g/L, the concentration of plasmid extraction is significantly higher than that of a culture medium with other concentrations of the same components. Especially, when the concentration of glucose is from 0.5 to 2 g/L, and the concentration of arabinose is from 0.3 to 0.75 g/L, compared with a conventional method for inducing a plasmid copy number, the concentration of plasmid extraction is increased by more than 45%, and compared with an induction method using a culture medium without glucose and arabinose, the concentration of plasmid extraction is increased by more than 310%. The culture medium plays an important role in inducing an increase in a plasmid copy number and achieving high-throughput production. 
     Explanation of Terms 
     As used herein, the term “plasmid” refers to a DNA molecule outside a chromosome (or a nucleoid) within organisms such as bacteria, yeasts, and actinomycetes. The plasmid exists in cytoplasm and can replicate independently, so that the plasmid can also maintain a constant copy number in progeny cells and express carried genetic information. The plasmid is a closed circular, double-stranded DNA molecule. 
     The term “pCCIBAC plasmid” refers to a single-copy bacterial artificial chromosome. 
     The term “plasmid copy number” refers to the number of plasmids in a genome of a specific organism. Under the control of a plasmid replicon, the plasmid copy number may fluctuate in a narrow range with changes of bacterial culture conditions. When growth conditions are constant, the speed of plasmid proliferation is exactly the same as the speed of host cell proliferation, and the copy number remains unchanged. 
     The term “single-copy plasmid” means that there is only one specific plasmid in a biological genome. 
     The term “competent” refers to a special physiological state in which a cell can take up DNA molecules from a surrounding environment and the DNA molecules are not easily broken down by restriction endonucleases in the cell. 
     The term “glucose” refers to a polyhydroxy aldehyde with the molecular formula C 6 H 12 O 6 , which is important in the field of biology and is an energy source for living cells and an intermediate product of metabolism. 
     The term “arabinose” is also referred to as L(+)-arabinose, pectinose, or the like, with the molecular formula C 5 H 10 O 5 , which is a levorotatory monosaccharide. 
     The term “tryptone” is also referred to as casein tryptone and pancreatic digest of casein, which is a high-quality peptone and is light yellow powder formed through concentration and drying. The tryptone is rich in nitrogen sources, amino acids and the like, can be used to prepare various microbial culture media for culture, isolation, proliferation, and identification of bacteria, and can be used to prepare bacterial biochemical characteristics test culture media such as a sterile test culture medium and an anaerobic bacteria culture medium. 
     The term “yeast extract” is also referred to as yeast flavor, which is abbreviated as YE according to international usage. Main components of the yeast extract are polypeptides, amino acids, 5′-ribonucleotide, B vitamins, and trace elements. The yeast extract is the most ideal raw material for biological culture media and the main raw material in fermentation industries. The yeast extract has the efficacy 8 times that of yeast, which can greatly increase the production rate of bacterial strains and the yield of fermentation products. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    shows a result of plasmid extraction in Example 1, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; 
         FIG.  2    shows a result of plasmid extraction in Example 2, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; 
         FIG.  3    shows a result of plasmid extraction in Example 3, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; 
         FIG.  4    shows a result of plasmid extraction in Example 4, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; 
         FIG.  5    shows a result of plasmid extraction in Example 5, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; 
         FIG.  6    shows a result of plasmid extraction in Example 6, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; 
         FIG.  7    shows a result of plasmid extraction in Comparative Example 1, where a horizontal coordinate is a sampling time, and a longitudinal coordinate is a concentration of extracted plasmids; and 
         FIG.  8    is a diagram of a pCCIBAC vector. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention is further described below with reference to the specific implementations. 
     Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. 
     Unless otherwise specified, the materials and reagents used in the following examples can be commercially available. 
     Example 1 
     The pCCIBAC plasmid (shown as SEQ ID NO: 1) included a replication origin oriV. The pCCIBAC plasmid was transformed into competent EPI300  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride, 2 g/L of glucose, and 0.75 g/L of arabinose. The bacteria were cultured at 220 r/min and 25° C. for 5 h. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  1   . 
     Example 2 
     The pCCIBAC plasmid included a replication origin oriV. The plasmid was transformed into competent EPI400  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 5 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of sodium chloride, 1 g/L of glucose, and 0.6 g/L of arabinose. The bacteria were cultured at 220 r/min and 30° C. for 5 h. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  2   . 
     Example 3 
     The pCCIBAC plasmid included a replication origin oriV. The pCCIBAC plasmid was transformed into competent EPI300  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 10 g/L of yeast extract, 10 g/L of sodium chloride, 0.5 g/L of glucose, and 0.3 g/L of arabinose. The bacteria were cultured at 220 r/min and 37° C. for 5 h. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  3   . 
     Example 4 
     The pCCIBAC plasmid included a replication origin oriV. The plasmid was transformed into competent EPI400  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of sodium chloride, 2 g/L of glucose, and 1 g/L of arabinose. The bacteria were cultured at 220 r/min and 30° C. for 5 h. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  4   . 
     Example 5 
     The pCCIBAC plasmid included a replication origin oriV. The plasmid was transformed into competent EPI400  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of sodium chloride, 10 g/L of glucose, and 5 g/L of arabinose. The bacteria were cultured at 220 r/min and 30° C. for 5 h. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  5   . 
     Example 6 
     The pCCIBAC plasmid included a replication origin oriV. The plasmid was transformed into competent EPI400  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of sodium chloride, 10 g/L of glucose, and 2 g/L of arabinose. The bacteria were cultured at 220 r/min and 30° C. for 5 h. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  6   . 
     Comparative Example 1 (without Glucose and Arabinose) 
     The pCCIBAC plasmid included a replication origin oriV. The pCCIBAC plasmid was transformed into competent EPI300  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 10 g/L of yeast extract, and 10 g/L of sodium chloride. The bacteria were cultured at 220 r/min and 30° C. After the inoculation, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  7   . 
     Comparative Example 2 (Conventional Method for Inducing a Plasmid Copy Number) 
     The pCCIBAC plasmid included a replication origin oriV. The pCCIBAC plasmid was transformed into competent EPI300  Escherichia coli  by chemical transformation. The transformed  Escherichia coli  was cultured in the LB culture medium (in which components were 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride) overnight, and then was inoculated at an inoculation rate of 5% into a culture medium in which components were 10 g/L of tryptone, 10 g/L of yeast extract, and 10 g/L of sodium chloride. The bacteria were cultured at 220 r/min and 30° C. for 12 h, and then, were used as a seed solution. Re-inoculation was performed with the seed solution, and OD600 after the re-inoculation was 0.2. Then, the bacteria were cultured at 30° C. for 1 h, and arabinose was added to make the concentration be 0.2 g/L. The culture was continued for 5 h, 6 OD600 bacteria were taken every 1 h for plasmid extraction. The plasmid extraction was performed by using the standard procedure of the plasmid extraction kit AxyGEN Miniprep Kit (Lot #17718KA1). The result of the plasmid extraction was shown in  FIG.  8   . 
     After the plasmid extraction, plasmid concentrations were measured by using Nano Drop oneC (Thermo Fisher Scientific). Results were shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Plasmid extraction concentration (ng/μL) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Time 
                 1 h 
                 2 h 
                 3 h 
                 4 h 
                 5 h 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Comparative 
                 12.5 
                 15.4 
                 13.2 
                 16.4 
                 16.4 
               
               
                   
                 Example 1 
               
               
                   
                 Example 1 
                 14.5 
                 20.2 
                 48.6 
                 55.4 
                 78.5 
               
               
                   
                 Example 2 
                 14.3 
                 15.4 
                 46.4 
                 74.5 
                 88.5 
               
               
                   
                 Example 3 
                 12.8 
                 13.3 
                 37.8 
                 62.3 
                 68.4 
               
               
                   
                 Example 4 
                 21.2 
                 18.3 
                 18.4 
                 35.6 
                 42.2 
               
               
                   
                 Example 5 
                 18.4 
                 15.3 
                 28.4 
                 34.1 
                 34.4 
               
               
                   
                 Example 6 
                 13.1 
                 17.4 
                 25.2 
                 33.8 
                 41.5 
               
               
                   
                 Comparative 
                 12.5 
                 24.8 
                 27.8 
                 38.3 
                 58.4 
               
               
                   
                 Example 2 
               
               
                   
                   
               
            
           
         
       
     
     The comparison of the plasmid extraction concentrations between Examples 1 to 6 and Comparative Example 1 was shown in Table 2. After glucose and arabinose were added into the culture medium, the concentration of the extracted plasmids increased rapidly. After culture in the culture medium for 5 h, the plasmid extraction concentrations increased by from 110 to 440%. Especially, when the concentration of glucose was from 0.5 to 2 g/L and the concentration of arabinose was from 0.3 to 0.75 g/L, after culture in the culture medium for 5 h, the plasmid extraction concentrations increased by from 317 to 440%. It indicates that the culture medium provided by the present invention is suitable for inducing an increase in a plasmid copy number and greatly improving the plasmid yield. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Percentages of increases of plasmid extraction concentrations 
               
               
                 of Examples 1 to 6 compared with Comparative Example 1 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Time 
                 1 h 
                 2 h 
                 3 h 
                 4 h 
                 5 h 
               
               
                   
                   
               
               
                   
                 Example 1 
                 16% 
                 31% 
                 268% 
                 238% 
                 379% 
               
               
                   
                 Example 2 
                 14% 
                  0% 
                 252% 
                 354% 
                 440% 
               
               
                   
                 Example 3 
                  2% 
                 −14%  
                 186% 
                 280% 
                 317% 
               
               
                   
                 Example 4 
                 70% 
                 19% 
                  39% 
                 117% 
                 157% 
               
               
                   
                 Example 5 
                 47% 
                 −1% 
                 115% 
                 108% 
                 110% 
               
               
                   
                 Example 6 
                  5% 
                 13% 
                  91% 
                 106% 
                 153% 
               
               
                   
                   
               
            
           
         
       
     
     Compared with Comparative Example 2 (the conventional method for inducing a plasmid copy number), Examples 1 to 6 had simple operations and greatly improved plasmid extraction concentrations. As shown in Table 3, when the concentration of glucose was from 0.5 to 2 g/L and the concentration of arabinose was from 0.3 to 0.75 g/L, after extraction for 3 h, the plasmid extraction concentrations increased by from 36 to 75% compared to the conventional method, and after extraction for 4 h, the plasmid extraction concentrations increased by from 45 to 95% compared to the conventional method. Especially, when the concentration of glucose was 1 g/L and the concentration of arabinose was 0.6 g/L, after extraction for 4 h, the plasmid extraction concentrations increased by 95% compared to the conventional method. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Percentages of increases of plasmid extraction concentrations 
               
               
                 of Examples 1 to 6 compared with Comparative Example 2 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Time 
                 1 h 
                 2 h 
                 3 h 
                 4 h 
                 5 h 
               
               
                   
                   
               
               
                   
                 Example 1 
                 16% 
                 −19% 
                 75% 
                 45% 
                  34% 
               
               
                   
                 Example 2 
                 14% 
                 −38% 
                 67% 
                 95% 
                  52% 
               
               
                   
                 Example 3 
                  2% 
                 −46% 
                 36% 
                 63% 
                  17% 
               
               
                   
                 Example 4 
                 70% 
                 −26% 
                 −34%  
                 −7% 
                 −28% 
               
               
                   
                 Example 5 
                 47% 
                 −38% 
                  2% 
                 −11%  
                 −41% 
               
               
                   
                 Example 6 
                  5% 
                 −30% 
                 −9% 
                 −12%  
                 −29% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
            
               
                 SEQ ID NO: 1 
                   
               
               
                 pCCIBAC vector sequence 
                   
               
               
                 gcggccgcaaggggttcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagc 
               
               
                   
               
               
                 agattgtactgagagtgcaccatatgcggtgtgaaataccacacagatgcgtaaggagaaaataccgcatcag 
               
               
                   
               
               
                 gcgccattcgccattcagctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct 
               
               
                   
               
               
                 ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaa 
               
               
                   
               
               
                 acgacggccagtgaattgtaatacgactcactatagggcgaattcgagctcggtacccggggatcctctagagt 
               
               
                   
               
               
                 cgacctgcaggcatgcaagcttgagtattctatagtctcacctaaatagcttggcgtaatcatggtcatagctgttt 
               
               
                   
               
               
                 cctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggt 
               
               
                   
               
               
                 gcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgcc 
               
               
                   
               
               
                 agctgcattaatgaatcggccaacgcgaaccccttgcggccgcccgggccgtcgaccaattctcatgtttgaca 
               
               
                   
               
               
                 gcttatcatcgaatttctgccattcatccgcttattatcacttattcaggcgtagcaaccaggcgtttaagggcacca 
               
               
                   
               
               
                 ataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccga 
               
               
                   
               
               
                 catggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataa 
               
               
                   
               
               
                 tatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactc 
               
               
                   
               
               
                 acccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaa 
               
               
                   
               
               
                 cacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaac 
               
               
                   
               
               
                 gtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgc 
               
               
                   
               
               
                 catacgaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatt 
               
               
                   
               
               
                 tttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaa 
               
               
                   
               
               
                 atgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagctt 
               
               
                   
               
               
                 ccttagctcctgaaaatctcgataactcaasaaatacgcccggtagtgatcttatttcattatggtgaaagttggaa 
               
               
                   
               
               
                 cctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagggacacca 
               
               
                   
               
               
                 ggatttatttattctgcgaagtgatcttccgtcacaggtatttattcgcgataagctcatggagcggcgtaaccgtc 
               
               
                   
               
               
                 gcacaggaaggacagagaaagcgcggatctgggaagtgacggacagaacggtcaggacctggattgggg 
               
               
                   
               
               
                 aggcggttgccgccgctgctgctgacggtgtgacgttctctgttccggtcacaccacatacgttccgccattcct 
               
               
                   
               
               
                 atgcgatgcacatgctgtatgccggtataccgctgaaagttctgcaaagcctgatgggacataagtccatcagtt 
               
               
                   
               
               
                 caacggaagtctacacgaaggtttttgcgctggatgtggctgcccggcaccgggtgcagtttgcgatgccgga 
               
               
                   
               
               
                 gtctgatgcggttgcgatgctgaaacaattatcctgagaataaatgccttggcctttatatggaaatgtggaactga 
               
               
                   
               
               
                 gtggatatgctgtttttgtctgttaaacagagaagctggctgttatccactgagaagcgaacgaaacagtcggga 
               
               
                   
               
               
                 aaatctcccattatcgtagagatccgcattattaatctcaggagcctgtgtagcgtttataggaagtagtgttctgtc 
               
               
                   
               
               
                 atgatgcctgcaagcggtaacgaaaacgatttgaatatgccttcaggaacaatagaaatcttcgtgcggtgttac 
               
               
                   
               
               
                 gttgaagtggagcggattatgtcagcaatggacagaacaacctaatgaacacagaaccatgatgtggtctgtcc 
               
               
                   
               
               
                 ttttacagccagtagtgctcgccgcagtcgagcgacagggcgaagccctcgagctggttgccctcgccgctgg 
               
               
                   
               
               
                 gctggcggccgtctatggccctgcaaacgcgccagaaacgccgtcgaagccgtgtgcgagacaccgcggc 
               
               
                   
               
               
                 cggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttgacac 
               
               
                   
               
               
                 ttgaggggccgactcacccggcgcggcgttgacagatgaggggcaggctcgatttcggccggcgacgtgga 
               
               
                   
               
               
                 gctggccagcctcgcaaatcggcgaaaacgcctgattttacgcgagtttcccacagatgatgtggacaagcctg 
               
               
                   
               
               
                 gggataagtgccctgcggtattgacacttgaggggcgcgactactgacagatgaggggcgcgatccttgaca 
               
               
                   
               
               
                 cttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgaggggctgtccacaggcagaaaa 
               
               
                   
               
               
                 tccagcatttgcaagggtttccgcccgtttttcggccaccgctaacctgtcttttaacctgcttttaaaccaatatttat 
               
               
                   
               
               
                 aaaccttgtttttaaccagggctgcgccctgtgcgcgtgaccgcgcacgccgaaggggggtgcccccccttct 
               
               
                   
               
               
                 cgaaccctcccggtcgagtgagcgaggaagcaccagggaacagcacttatatattctgcttacacacgatgcc 
               
               
                   
               
               
                 tgaaasaacttcccttggggttatccacttatccacggggatatttttataattattttttttatagtttttagatcttcttttt 
               
               
                   
               
               
                 tagagcgccttgtaggcctttatccatgctggttctagagaaggtgttgtgacaaattgccctttcagtgtgacaaa 
               
               
                   
               
               
                 tcaccctcaaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctagaagaagctgttt 
               
               
                   
               
               
                 tttcacaaagttatccctgcttattgactcttttttatttagtgtgacaatctaaaaacttgtcacacttcacatggatctg 
               
               
                   
               
               
                 tcatggcggaaacagcggttatcaatcacaagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacct 
               
               
                   
               
               
                 cactgaggcggcatatagtctctcccgggatcaaaaacgtatgctgtatctgttcgttgaccagatcagaaaatct 
               
               
                   
               
               
                 gatggcaccctacaggaacatgacggtatctgcgagatccatgttgctaaatatgctgaaatattcggattgacct 
               
               
                   
               
               
                 ctgcggaagccagtaaggatatacggcaggcattgaagagtttcgcggggaaggaagtggttttttatcgccct 
               
               
                   
               
               
                 gaagaggatgccggcgatgaaaaaggctatgaatcttttccttggtttatcaaacgtgcgcacagtccatccaga 
               
               
                   
               
               
                 gggctttacagtgtacatatcaacccatatctcattcccttctttatcgggttacagaaccggtttacgcagtttcgg 
               
               
                   
               
               
                 cttagtgaaacaaaagaaatcaccaatccgtatgccatgcgtttatacgaatccctgtgtcagtatcgtaagccgg 
               
               
                   
               
               
                 atggctcaggcatcgtctctctgaaaatcgactggatcatagagcgttaccagctgcctcaaagttaccagcgta 
               
               
                   
               
               
                 tgcctgacttccgccgccgcttcctgcaggtctgtgttaatgagatcaacagcagaactccaatgcgcctctcata 
               
               
                   
               
               
                 cattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacagg 
               
               
                   
               
               
                 atagtctgagggttatctgtcacagatttgagggtggttcgtcacatttgttctgacctactgagggtaatttgtcac 
               
               
                   
               
               
                 agttttgctgtttccttcagcctgcatggattttctcatactttttgaactgtaatttttaaggaagccaaatttgagggc 
               
               
                   
               
               
                 agtttgtcacagttgatttccttctctttcccttcgtcatgtgacctgatatcgggggttagttcgtcatcattgatgag 
               
               
                   
               
               
                 ggttgattatcacagtttattactctgaattggctatccgcgtgtgtacctctacctggagtttttcccacggtggata 
               
               
                   
               
               
                 tttcttcttgcgctgagcgtaagagctatctgacagaacagttcttctttgcttcctcgccagttcgctcgctatgctc 
               
               
                   
               
               
                 ggttacacggctgcggcgagcgctagtgataataagtgactgaggtatgtgctcttcttatctccttttgtagtgttg 
               
               
                   
               
               
                 ctcttattttaaacaactttgcggttttttgatgactttgcgattttgttgttgctttgcagtaaattgcaagatttaataaa 
               
               
                   
               
               
                 aaaacgcaaagcaatgattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgcataaacgctgg 
               
               
                   
               
               
                 tcatgaaatgacgaaggctatcgccattgcacagtttaatgatgacagcccggaagcgaggaasataacccgg 
               
               
                   
               
               
                 cgctggagaataggtgaagcagcggatttagttggggtttcttctcaggctatcagagatgccgagaaagcagg 
               
               
                   
               
               
                 gcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggttatacaattgaacaaatta 
               
               
                   
               
               
                 atcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttgctgc 
               
               
                   
               
               
                 ccataaaggtggcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgtttt 
               
               
                   
               
               
                 gctcgtggaaggtaacgacccccagggaacagcctcaatgtatcacggatgggtaccagatcttcatattcatg 
               
               
                   
               
               
                 cagaagacactctcctgcctttctatcttggggaaaaggacgatgtcacttatgcaataaagcccacttgctggcc 
               
               
                   
               
               
                 ggggcttgacattattccttcctgtctggctctgcaccgtattgaaactgagttaatgggcaaatttgatgaaggta 
               
               
                   
               
               
                 aactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtcatagttatt 
               
               
                   
               
               
                 gacagcgcgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctg 
               
               
                   
               
               
                 ctgagttgtttgactacacctccgcactgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaag 
               
               
                   
               
               
                 ggttcgagcctgatgtacgtattttgcttaccaaatacagcaatagcaatggctctcagtccccgtggatggagg 
               
               
                   
               
               
                 agcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacgtgaaacggatgaagttggtaaaggt 
               
               
                   
               
               
                 cagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggagaaatgctcttt 
               
               
                   
               
               
                 ctatttgggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtg 
               
               
                   
               
               
                 cgcctgttattccaaaacatacgctcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgat 
               
               
                   
               
               
                 ggtggattcgttaattgcgcgcgtaggagtaatggctcgcggtaatgccattactttgcctgtatgtggtcgggat 
               
               
                   
               
               
                 gtgaagtttactcttgaagtgctccggggtgatagtgttgagaagacctctcgggtatggtcaggtaatgaacgt 
               
               
                   
               
               
                 gaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtcaacagacaccg 
               
               
                   
               
               
                 gcgttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttacc 
               
               
                   
               
               
                 gaaagtgattatcgtgttctggttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgatt 
               
               
                   
               
               
                 atcgcccaacaagtgcttatgaacgtggtcagcgttatgcaagccgattgcagaatgaatttgctggaaatatttc 
               
               
                   
               
               
                 tgcgctggctgatgcggaaaatatttcacgtaagattattacccgctgtatcaacaccgccaaattgcctaaatca 
               
               
                   
               
               
                 gttgttgctcttttttctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaaaagcctttacagataaa 
               
               
                   
               
               
                 gaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaaga 
               
               
                   
               
               
                 agttatcactcttttaacttctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctc 
               
               
                   
               
               
                 ctggagcgacagtattgtataagggcgataaaatggtgcttaacctggacaggtctcgtgttccaactgagtgtat 
               
               
                   
               
               
                 agagaaaattgaggccattcttaaggaacttgaaaagccagcaccctgatgcgaccacgttttagtctacgtttat 
               
               
                   
               
               
                 ctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaatattctctctgggcccactgttcca 
               
               
                   
               
               
                 cttgtatcgtcggtctgataatcagactgggaccacggtcccactcgtatcgtcggtctgattattagtctgggacc 
               
               
                   
               
               
                 acggtcccactcgtatcgtcggtctgattattagtctcggaccacggtcccactcgtatcgtcggtctgataatca 
               
               
                   
               
               
                 gactgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccatggtcccactcgtatcgtcggt 
               
               
                   
               
               
                 ctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctggaaccacggtcccactcgt 
               
               
                   
               
               
                 atcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacgat 
               
               
                   
               
               
                 cccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagcgtga 
               
               
                   
               
               
                 gactacgattccatcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcg 
               
               
                   
               
               
                 gtgtgcggttgtatgcctgctgtggattgctgctgtgtcctgcttatccacaacattttgcgcacggttatgtggaca 
               
               
                   
               
               
                 aaatacctggttacccaggccgtgccggcacgttaaccgggctgcatccgatgcaagtgtgtcgctgtcgacg 
               
               
                   
               
               
                 agctcgcgagctcggacatgaggttgccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaa 
               
               
                   
               
               
                 gttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccacgcacgttg 
               
               
                   
               
               
                 tgatatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcg 
               
               
                   
               
               
                 cgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaat 
               
               
                   
               
               
                 accctctgaaaagaaaggaaacgacaggtgctgaaagcgagctttttggcctctgtcgtttcctttctctgtttttgt 
               
               
                   
               
               
                 ccgtggaatgaacaatggaagtccgagctcatcgctaataacttcgtatagcatacattatacgaagttatattcg 
               
               
                   
               
               
                 at 
               
            
           
         
       
     
     The culture medium for inducing an increase in a plasmid copy number and use thereof provided in the present invention are described in detail above. The principle and implementations of the present invention are described herein through specific examples. The description about the foregoing examples is merely provided to help understand the method and core ideas of the present invention. It should be noted that a person skilled in the art may further make several improvements and modifications to the present invention without departing from the principle of the present invention. These improvements and modifications also fall within the protection scope of the claims of the present invention.