WO2020135549A1 - 诱导质粒拷贝数增加的培养基及其应用 - Google Patents

诱导质粒拷贝数增加的培养基及其应用 Download PDF

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WO2020135549A1
WO2020135549A1 PCT/CN2019/128523 CN2019128523W WO2020135549A1 WO 2020135549 A1 WO2020135549 A1 WO 2020135549A1 CN 2019128523 W CN2019128523 W CN 2019128523W WO 2020135549 A1 WO2020135549 A1 WO 2020135549A1
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plasmid
medium
copy number
concentration
inducing
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French (fr)
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赵方龙
马艳秋
王嫚
刘凡
吴政宪
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江苏金斯瑞生物科技有限公司
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Priority to US17/417,802 priority Critical patent/US20230159940A1/en
Priority to EP19901649.4A priority patent/EP3885430A4/en
Publication of WO2020135549A1 publication Critical patent/WO2020135549A1/zh

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  • the present invention belongs to the field of biotechnology, and specifically relates to a medium for inducing an increase in plasmid copy number and its application.
  • the present invention provides a medium for inducing an increase in plasmid copy number and its application, which simplifies operation and improves production efficiency.
  • An aspect of the present invention provides a medium for inducing an increase in plasmid copy number, characterized in that the medium contains tryptone, yeast extract, glucose, sodium chloride, and arabinose.
  • the glucose concentration is 0.1-10 g/L, preferably 1-5 g/L, and more preferably 0.5-2 g/L.
  • the concentration of glucose is 0.1-10 g/L. In some embodiments, the concentration of glucose is 1-5 g/L. In some embodiments, the glucose concentration is 0.5-2 g/L. In some embodiments, the glucose concentration is 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L , 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L, 2g/L. In some embodiments, the concentration of glucose is 0.5 g/L. In some embodiments, the glucose concentration is 1 g/L. In some embodiments, the glucose concentration is 2 g/L. In some embodiments, the concentration of glucose is 10 g/L.
  • the concentration of arabinose is 0.1-10 g/L, preferably 1-5 g/L, and more preferably 0.3-5 g/L.
  • the concentration of arabinose is 0.1-10 g/L. In some embodiments, the concentration of arabinose is 1-5 g/L. In some embodiments, the concentration of arabinose is 0.3-0.75 g/L. In some embodiments, the concentration of arabinose is 0.3g/L, 0.35g/L, 0.4g/L, 0.45g/L, 0.5g/L, 0.55g/L, 0.6g/L, 0.65g /L, 0.7g/L, 0.75g/L. In some embodiments, the concentration of arabinose is 0.3 g/L. In some embodiments, the concentration of arabinose is 0.6 g/L. In some embodiments, the concentration of arabinose is 0.75 g/L. In some embodiments, the concentration of arabinose is 1 g/L. In some embodiments, the concentration of arabinose is 2 g/L. In some embodiments, the concentration of arabinose is 5 g/L.
  • the concentration of tryptone is 1-15 g/L.
  • the concentration of tryptone is 5-10 g/L.
  • the concentration of the yeast extract is 1-15 g/L.
  • the concentration of the yeast extract is 5-10 g/L.
  • the concentration of sodium chloride is 1-15 g/L.
  • the concentration of sodium chloride is 5-10 g/L.
  • the medium further contains one or more of magnesium chloride, potassium chloride, ferric chloride or calcium chloride.
  • the plasmid is a plasmid containing an origin of oriV replication.
  • the plasmid is a single copy plasmid containing an oriV replication initiation site.
  • the plasmid is a single copy pCCIBAC plasmid.
  • Another aspect of the invention provides a method for inducing an increase in plasmid copy number, including the following steps:
  • Step (1) Transfer the plasmid into E. coli;
  • Step (2) Inoculate the E. coli obtained in step (1) into the medium, and culture under suitable culture conditions.
  • the plasmid is transformed into E. coli competent in the step (1).
  • the E. coli in step (2) is preferably EPI300 E. coli or EPI400 E. coli.
  • the cultivation temperature in step (2) is 20-37°C.
  • the cultivation temperature in step (2) is 25-37°C.
  • the cultivation time in step (2) is 4-6h.
  • a single copy plasmid containing the origin of replication oriV is transformed into an E. coli strain, and in step (2), at a concentration of 1-5 g/L containing glucose, In the medium with arabinose concentration of 1-5g/L, tryptone concentration of 1-15g/L, yeast extract concentration of 1-15g/L and sodium chloride concentration of 1-15g/L, E. coli is cultured at 20-37°C.
  • step (1) the pCCIBAC plasmid is transformed into EPI300 E. coli or EPI400 E. coli.
  • step (2) the concentration of glucose containing 0.5-2g/L, arabinose In a medium with a concentration of 0.3-0.75g/L, a tryptone concentration of 5-10g/L, a yeast extract concentration of 5-10g/L, and a NaCl concentration of 5-10g/L, E. coli Culture at 25-37°C.
  • the use of the medium in inducing an increase in plasmid copy number is provided.
  • the invention provides a new culture medium, which can induce the increase of plasmid copy number and improve the plasmid yield.
  • the medium is used to induce the increase of plasmid copy number, the operation steps are simple, the cultivation time is greatly shortened, and there is no need for multi-step operations such as transfer, OD600 quantification and addition of inducer.
  • the concentration of plasmid extraction is significantly higher than that of other media of the same component, especially in the concentration of glucose of 0.5-2g/L
  • the concentration of arabinose is 0.3-0.75g/L
  • the plasmid extraction concentration is increased by more than 45%, compared to the medium and glucose-free and arabinose-free medium induction method, the plasmid
  • the extraction concentration is increased by more than 310%, and the medium plays an important role in inducing an increase in plasmid copy number and achieving high-throughput production.
  • plasmid refers to DNA molecules other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts, and actinomycetes, which exist in the cytoplasm and have the ability to autonomously replicate so that they can also be used in progeny cells Keeping a constant copy number and expressing the genetic information it carries is a closed circular double-stranded DNA molecule.
  • pCCIBAC plasmid refers to a single copy of a bacterial artificial chromosome.
  • plasmid copy number refers to the number of plasmids in the genome of an organism. Under the control of plasmid replicons, the plasmid copy number can fluctuate within a narrow range with changes in bacterial culture conditions. When the growth conditions are constant, the rate of plasmid proliferation is exactly the same as the rate of host cell proliferation, and the copy number remains unchanged.
  • single copy plasmid means that there is only one plasmid in the genome of an organism.
  • Competent refers to a special physiological state in which cells can take up DNA molecules from the surrounding environment and are not easily broken down by restriction endonuclease in the cell.
  • glucose refers to a polyhydroxy aldehyde with a molecular formula of C 6 H 12 O 6. It has an important position in the field of biology and is an energy source for living cells and an intermediate product of metabolism.
  • aromaticnose is also known as L(+)-gum aldose, L(+)-pentanose, pectinose, etc., with a molecular formula of C 5 H 10 O 5 , and is a levorotatory monosaccharide.
  • tryptone is also called casein peptone (Casein Tryptone), pancreatin digestion casein peptone (Pancreatic digest of casein), is a high-quality peptone, concentrated and dried light yellow powder. It is rich in nitrogen source, amino acid, etc. It can be used to prepare various microbial culture media for the cultivation, isolation, propagation and identification of bacteria, as well as culture media for bacterial biochemical characteristics tests such as sterile test media and anaerobic bacteria media. Configuration.
  • yeast extract is also known as yeast taste, which is abbreviated as YE according to international common usage.
  • the main components are peptides, amino acids, taste nucleotides, B vitamins and trace elements. It is the most ideal raw material for biological media and the main raw material in the fermentation industry. Its efficacy is equivalent to 8 times of yeast, which can greatly improve the strain Production rate and yield of fermented products.
  • Figure 1 shows the results of plasmid extraction in Example 1.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Figure 2 shows the results of plasmid extraction in Example 2.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Figure 3 shows the results of plasmid extraction in Example 3.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Figure 4 shows the results of plasmid extraction in Example 4.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Figure 5 shows the results of plasmid extraction in Example 5.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Figure 6 shows the results of plasmid extraction in Example 6.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Fig. 7 shows the results of plasmid extraction of Comparative Example 1.
  • the abscissa is the sampling time, and the ordinate is the extracted plasmid concentration;
  • Figure 8 shows a schematic representation of the pCCIBAC vector.
  • the pCCIBAC plasmid (as shown in SEQ ID NO: 1) contains the oriV replication start site.
  • the pCCIBAC plasmid was transformed into EPI300 E. coli competent cells by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and then inoculated into 10g/L tryptone, 5g/L In the medium of yeast extract, 10g/L sodium chloride, 2g/L glucose and 0.75g/L arabinose, the inoculation ratio is 5%.
  • the bacteria were cultured at 220 rpm for 5 hours at 25°C.
  • Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 1.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the plasmid was transformed into EPI400 E. coli competent state by chemical transformation.
  • the transformed E. coli was cultured overnight in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) and inoculated into 5g/L tryptone, 10g/L.
  • LB medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride
  • the inoculation ratio is 5%.
  • the bacteria were cultured at 220 rpm for 5 hours at 30°C. After inoculation, 6 OD600 cells were taken every 1h for plasmid extraction. Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 2.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the pCCIBAC plasmid was transformed into EPI300 E. coli competent cells by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and inoculated into 10g/L tryptone, 10g/L.
  • the bacteria were incubated at 220 rpm for 5 hours at 37°C. After inoculation, 6 OD600 cells were taken every 1h for plasmid extraction. Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 3.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the plasmid was transformed into EPI400 E. coli competent state by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and inoculated into 10g/L tryptone, 10g/L.
  • the bacteria were cultured at 220 rpm for 5 hours at 30°C. After inoculation, 6 OD600 cells were taken every 1h for plasmid extraction. Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 4.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the plasmid was transformed into EPI400 E. coli competent state by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and inoculated into 10g/L tryptone, 10g/L.
  • LB medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride
  • the inoculation ratio is 5%.
  • the bacteria were cultured at 220 rpm for 5 hours at 30°C. After inoculation, 6 OD600 cells were taken every 1h for plasmid extraction. Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 5.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the plasmid was transformed into EPI400 E. coli competent state by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and inoculated into 10g/L tryptone, 10g/L.
  • the bacteria were cultured at 220 rpm for 5 hours at 30°C. After inoculation, 6 OD600 cells were taken every 1h for plasmid extraction. Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 6.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the pCCIBAC plasmid was transformed into EPI300 E. coli competent cells by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and inoculated into 10g/L tryptone, 10g/L. In the medium of yeast extract and 10g/L sodium chloride, the inoculation ratio is 5%.
  • the bacteria were cultured at 220 rpm at 30°C. After inoculation, 6 OD600 cells were taken every 1h for plasmid extraction. Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). The results of plasmid extraction are shown in Figure 7.
  • the pCCIBAC plasmid contains the oriV replication start site.
  • the pCCIBAC plasmid was transformed into EPI300 E. coli competent cells by chemical transformation.
  • the transformed E. coli was cultured in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride) overnight, and inoculated into 10g/L tryptone, 10g/L.
  • the inoculation ratio is 5%.
  • the bacteria were cultured at 220 rpm for 12 hours at 30°C as seed fluid. The seed solution was inoculated again, and the OD600 was 0.2 after inoculation.
  • Plasmid extraction was performed using the standard procedure of plasmid extraction kit AxyGEN Miniprep Kit (Lot#17718KA1). See Table 1 for plasmid extraction results.
  • 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 Example 2 12.5 24.8 27.8 38.3 58.4
  • the concentration of the extracted plasmid increased rapidly.
  • the plasmid The extraction concentration is increased by 110-440%, especially when the concentration of glucose is 0.5-2g/L and the concentration of arabinose is 0.3-0.75g/L.
  • the concentration of plasmid extraction increases 317-440%, indicating that the medium provided by the present invention is suitable for inducing an increase in plasmid copy number and a greatly improved plasmid yield.
  • 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%
  • Examples 1-6 Compared with Comparative Example 2 (traditional plasmid copy number induction method), Examples 1-6 have a simple operation process, and the plasmid extraction concentration is greatly increased. As shown in Table 3, when the concentration of glucose is 0.5-2g/L and the concentration of arabinose is 0.3-0.75g/L, after 3 hours of extraction, the plasmid extraction concentration is increased by 36-75 compared with the traditional method %; after 4 hours of extraction, the plasmid extraction concentration is increased by 45-95% compared with the traditional method, especially when the concentration of glucose is 1g/L and the concentration of arabinose is 0.6g/L, the extraction is 4 hours After that, the concentration of plasmid extraction increased by 95% compared with the traditional method.
  • 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%

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Abstract

一种诱导质粒拷贝数增加的培养基及其应用，该培养基相比于传统质粒拷贝数诱导方法，质粒抽提浓度提高了45-95％，相比于不加葡萄糖与阿拉伯糖的培养基诱导方法，质粒抽提浓度提高了110-440％，该培养基在诱导质粒拷贝数增加以及实现高通量生产中起到很重要的作用。

Description

诱导质粒拷贝数增加的培养基及其应用
本申请要求于2018年12月27日提交中国专利局、申请号为201811610822.6、发明名称为“诱导质粒拷贝数增加的培养基及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及诱导质粒拷贝数增加的培养基及其应用。
背景技术
在基因合成领域，约有10％以上的基因序列会对宿主大肠杆菌产生毒性。这些毒性通常是由异源基因导入宿主表达的蛋白对宿主生理代谢产生了影响，进而影响宿主生长。为了抵御毒性基因，宿主大肠杆菌倾向于保留基因突变或者基因缺失的质粒，导致基因和质粒的稳定性降低。在生产中采用低拷贝的质粒能有效降低毒性基因的表达，同时提高宿主的生长活力和基因的稳定性。但是，低拷贝质粒产率较低，抽提较为困难，影响生产效率。为了解决这一问题，在质粒抽提前，需要将低拷贝质粒快速诱导至高拷贝可提高质粒产率。而现有诱导质粒拷贝数增加的方法需要转接、OD600定量及诱导剂添加等多步操作，较为复杂，难以满足实际高通量生产。
发明内容
本发明针对现有质粒拷贝数诱导系统操作繁琐的问题，提供了一种诱导质粒拷贝数增加的培养基及其应用，在简化操作的同时提高了生产效率。
本发明一方面提供了一种诱导质粒拷贝数增加的培养基，其特征在于，所述培养基包含胰蛋白胨、酵母提取物、葡萄糖、氯化钠和阿拉伯糖。
在一些实施方案中，所述葡萄糖的浓度为0.1-10g/L，优选为1-5g/L，更优选为0.5-2g/L。
在一些实施方案中，所述葡萄糖的浓度为0.1-10g/L。在一些实施方案中，所述葡萄糖的浓度为1-5g/L。在一些实施方案中，所述葡萄糖的浓度为0.5-2g/L。在一些实施方案中，所述葡萄糖的浓度为0.5g/L、0.6g/L、0.7g/L、0.8g/L、0.9g/L、1g/L、1.1g/L、1.2g/L、1.3g/L、1.4g/L、1.5g/L、1.6g/L、1.7g/L、1.8g/L、1.9g/L、2g/L。在一些实施方案中，所述葡萄糖的浓度为0.5g/L。在一些实施方案中，所述葡萄糖的浓度为1g/L。在一些实施方案中，所述葡萄糖的浓度为2g/L。在一些实施方案中，所述葡萄糖的浓度为10g/L。
在另一些实施方案中，所述阿拉伯糖的浓度为0.1-10g/L，优选为1-5g/L，更优选为0.3-5g/L。
在另一些实施方案中，所述阿拉伯糖的浓度为0.1-10g/L。在一些实施方案中，所述阿拉伯糖的浓度为1-5g/L。在一些实施方案中，所述阿拉伯糖的浓度为0.3-0.75g/L。在一些实施方案中，所述阿拉伯糖的浓度为0.3g/L、0.35g/L、0.4g/L、0.45g/L、0.5g/L、0.55g/L、0.6g/L、0.65g/L、0.7g/L、0.75g/L。在一些实施方案中，所述阿拉伯糖的浓度为0.3g/L。在一些实施方案中，所述阿拉伯糖的浓度为0.6g/L。在一些实施方案中，所述阿拉伯糖的浓度为0.75g/L。在一些实施方案中，所述阿拉伯糖的浓度为1g/L。在一些实施方案中，所述阿拉伯糖的浓度为2g/L。在一些实施方案中，所述阿拉伯糖的浓度为5g/L。
在又一些实施方案中，所述胰蛋白胨的浓度为1-15g/L。
在又一些优选实施方案中，所述胰蛋白胨的浓度为5-10g/L。
在又一些实施方案中，所述酵母提取物的浓度为1-15g/L。
在又一些优选实施方案中，所述酵母提取物的浓度为5-10g/L。
在又一些实施方案中，所述氯化钠的浓度为1-15g/L。
在又一些优选实施方案中，所述氯化钠的浓度为5-10g/L。
在一些更优选实施方案中，所述培养基中还包含有氯化镁、氯化钾、氯化铁或氯化钙中的一种或多种。
在一些实施方案中，所述质粒为含有oriV复制起始位点的质粒。
在一些实施方案中，所述质粒为含有oriV复制起始位点的单拷贝质 粒。
在一些实施方案中，所述质粒为单拷贝的pCCIBAC质粒。
本发明另一方面提供了一种诱导质粒拷贝数增加的方法，包括以下步骤：
步骤(1)：将质粒转入大肠杆菌中；
步骤(2)：将步骤(1)得到的大肠杆菌接种到所述培养基中，在适于培养条件下进行培养。
在一些实施方案中，所述步骤(1)中将质粒转入大肠杆菌感受态中。
在另一些实施方案中，所述步骤(2)中的大肠杆菌优选为EPI300大肠杆菌或EPI400大肠杆菌。
在另一些实施方案中，所述步骤(2)中的培养温度为20-37℃。
在另一些实施方案中，所述步骤(2)中的培养温度为25-37℃。
在另一些实施方案中，所述步骤(2)中的培养时间为4-6h。
在一些具体实施方案中，在步骤(1)中，将含有复制起始位点oriV的单拷贝质粒转入大肠杆菌菌株中，步骤(2)中，在包含葡萄糖的浓度1-5g/L、阿拉伯糖的浓度为1-5g/L、胰蛋白胨浓度为1-15g/L、酵母提取物的浓度为1-15g/L和氯化钠的浓度为1-15g/L的培养基中，将大肠杆菌在20-37℃条件下培养。
在另一些具体实施方案中，在步骤(1)中，将pCCIBAC质粒转入EPI300大肠杆菌或EPI400大肠杆菌中，步骤(2)中，在包含葡萄糖的浓度为0.5-2g/L、阿拉伯糖的浓度为0.3-0.75g/L、胰蛋白胨的浓度为5-10g/L、酵母提取物的浓度为5-10g/L和NaCl的浓度为5-10g/L的培养基中，将大肠杆菌在25-37℃条件下培养。
本发明再一方面提供了所述的培养基在诱导质粒拷贝数增加中的应用。
本发明提供了一种新型培养基，可以诱导质粒拷贝数增加，提高质粒产率。同时，该培养基用于诱导质粒拷贝数增加时操作步骤简单，培养时间大幅缩短，且无需转接、OD600定量及诱导剂添加等多步操作。在葡萄糖的浓度低于2g/L且阿拉伯糖的浓度低于0.75g/L时，质粒抽提浓度 明显高于同组份其他浓度的培养基，尤其是在葡萄糖的浓度为0.5-2g/L且阿拉伯糖的浓度为0.3-0.75g/L时，相比于传统质粒拷贝数诱导方法，质粒抽提浓度提高了45％以上，相比于不含葡萄糖和阿拉伯糖的培养基诱导方法，质粒抽提浓度提高了310％以上，该培养基在诱导质粒拷贝数增加以及实现高通量生产中起到很重要的作用。
术语解释
如本文所用，术语“质粒”指细菌、酵母菌和放线菌等生物中染色体(或拟核)以外的DNA分子，存在于细胞质中，具有自主复制能力，使其在子代细胞中也能保持恒定的拷贝数，并表达所携带的遗传信息，是闭合环状的双链DNA分子。
术语“pCCIBAC质粒”指一种单拷贝的细菌人工染色体。
术语“质粒拷贝数”是指质粒在某一生物的基因组中的个数。在质粒复制子的调控下，质粒拷贝数可随细菌培养条件的变化在一个较窄的范围内波动。生长条件恒定时，质粒增殖的速度与宿主细胞增殖的速度完全一致，拷贝数保持不变。
术语“单拷贝质粒”指某一质粒在生物基因组中只有一个。
术语“感受态”指细胞能够从周围环境中摄取DNA分子，并且不易被细胞内的限制性核酸内切酶分解时所处的一种特殊生理状态。
术语“葡萄糖”是指一种多羟基醛，分子式为C 6H 12O 6，在生物学领域具有重要地位，是活细胞的能量来源和新陈代谢的中间产物。
术语“阿拉伯糖”又被称为L(+)-树胶醛糖、L(+)-阿戊糖、果胶糖等，分子式C 5H 10O 5，是一种左旋单糖。
术语“胰蛋白胨”又称胰酪蛋白胨(Casein Tryptone)、胰酶消化酪蛋白胨(Pancreatic digest of casein)，是一种优质蛋白胨，浓缩干燥而成的浅黄色粉末。含有丰富的氮源、氨基酸等，可配制各种微生物培养基，用于细菌的培养、分离、增殖、鉴定，以及无菌试验培养基、厌氧菌培养基等细菌生化特性试验用培养基的配置。
术语“酵母提取物”又称酵母味素，根据国际通用用法，将其缩写为YE。主要成分为多肽、氨基酸、呈味核苷酸、B族维生素及微量元素， 是最为理想的生物培养基原料和发酵工业中的主要原料，其功效与8倍的酵母相当，可以大大提高菌种的生产速率及发酵产品得率。
附图说明
图1示实施例1质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图2示实施例2质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图3示实施例3质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图4示实施例4质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图5示实施例5质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图6示实施例6质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图7示对比实施例1质粒抽提结果。横坐标为取样时间，纵坐标为抽提的质粒浓度；
图8示pCCIBAC载体图示。
具体实施方式
下面通过具体实施方式对本发明做进一步的说明。
下面实施例中所用实验方法如无特殊说明，均为常规方法。
下述实施例中所用材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
pCCIBAC质粒(如SEQ ID NO：1所示)含有oriV复制起始位点。将pCCIBAC质粒采用化学转化转入EPI300大肠杆菌感受态中。转化后 的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，2g/L葡萄糖和0.75g/L阿拉伯糖的培养基中，接种比例为5％。细菌在220转/分钟，25℃条件下培养5小时。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图1。
实施例2
pCCIBAC质粒含有oriV复制起始位点。将质粒采用化学转化转入EPI400大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为5g/L胰蛋白胨，10g/L酵母提取物，5g/L氯化钠，1g/L葡萄糖和0.6g/L阿拉伯糖的培养基中，接种比例为5％。细菌在220转/分钟，30℃条件下培养5小时。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图2。
实施例3
pCCIBAC质粒含有oriV复制起始位点。将pCCIBAC质粒采用化学转化转入EPI300大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，10g/L酵母提取物，10g/L氯化钠，0.5g/L葡萄糖和0.3g/L阿拉伯糖的培养基中，接种比例为5％。细菌在220转/分钟，37℃条件下培养5小时。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图3。
实施例4
pCCIBAC质粒含有oriV复制起始位点。将质粒采用化学转化转入EPI400大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，10g/L酵母提取物，5g/L氯化钠，2g/L葡萄糖和1g/L阿拉伯糖的培养基中，接种比例为5％。细菌在220转/分钟，30℃条件下培养5小时。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图4。
实施例5
pCCIBAC质粒含有oriV复制起始位点。将质粒采用化学转化转入EPI400大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，10g/L酵母提取物，5g/L氯化钠，10g/L葡萄糖和5g/L阿拉伯糖的培养基中，接种比例为5％。细菌在220转/分钟，30℃条件下培养5小时。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图5。
实施例6
pCCIBAC质粒含有oriV复制起始位点。将质粒采用化学转化转入EPI400大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，10g/L酵母提取物，5g/L氯化钠，10g/L葡萄糖和2g/L阿拉伯糖的培养基中，接种比例为5％。细菌在220转/分钟，30℃条件下培养5小时。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图6。
对比实施例1(不含葡萄糖和阿拉伯糖)
pCCIBAC质粒含有oriV复制起始位点。将pCCIBAC质粒采用化学转化转入EPI300大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，10g/L酵母提取物和10g/L氯化钠的培养基中，接种比例为5％。细菌在220转/分钟，30℃条件下培养。接菌后，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见图7。
对比实施例2(传统质粒拷贝数诱导方法)
pCCIBAC质粒含有oriV复制起始位点。将pCCIBAC质粒采用化学转化转入EPI300大肠杆菌感受态中。转化后的大肠杆菌在LB培养基(成分为10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠)中过夜培养后，接种于成分为10g/L胰蛋白胨，10g/L酵母提取物和10g/L氯化钠的培养基中，接种比例为5％。细菌在220转/分钟，30℃条件下培养12h后作为种子液。以此种子液重新接菌，接菌后OD600为0.2。继续在30℃条件下培养细菌1h，加入阿拉伯糖，使浓度为0.2g/L。继续培养5h，每隔1h取6个OD600的菌体用于质粒抽提。质粒抽提采用质粒抽提试剂盒AxyGEN Miniprep Kit(Lot#17718KA1)的标准流程进行抽提。质粒抽提结果见表1。
质粒抽提后，用Nano Drop oneC(Thermo Fisher Scientific)测定质粒浓度。结果如表1所示。
表1质粒抽提浓度(ng/uL)
时间 1h 2h 3h 4h 5h
对比实施例1 12.5 15.4 13.2 16.4 16.4
实施例1 14.5 20.2 48.6 55.4 78.5
实施例2 14.3 15.4 46.4 74.5 88.5
实施例3 12.8 13.3 37.8 62.3 68.4
实施例4 21.2 18.3 18.4 35.6 42.2
实施例5 18.4 15.3 28.4 34.1 34.4
实施例6 13.1 17.4 25.2 33.8 41.5
对比实施例2 12.5 24.8 27.8 38.3 58.4
实施例1-6与对比实施例1的质粒抽提浓度相比如表2结果所示，培养基中加入葡萄糖和阿拉伯糖后，抽提质粒浓度快速提高，在培养基中培养5小时后，质粒抽提浓度提高了110-440％，尤其是在葡萄糖的浓度在0.5-2g/L且阿拉伯糖的浓度在0.3-0.75g/L时，在培养基中培养5小时后，质粒抽提浓度提高了317-440％，表明本发明提供的培养基适用于诱导质粒拷贝数增加且质粒产率大大提高。
表2实施例1-6与对比实施例1相比质粒抽提浓度提高百分比
时间 1h 2h 3h 4h 5h
实施例1 16％ 31％ 268％ 238％ 379％
实施例2 14％ 0％ 252％ 354％ 440％
实施例3 2％ -14％ 186％ 280％ 317％
实施例4 70％ 19％ 39％ 117％ 157％
实施例5 47％ -1％ 115％ 108％ 110％
实施例6 5％ 13％ 91％ 106％ 153％
实施例1-6与对比实施例2(传统质粒拷贝数诱导方法)相比，操作过程简单，质粒抽提浓度大幅提高。如表3所示，在葡萄糖的浓度在0.5-2g/L且阿拉伯糖的浓度在0.3-0.75g/L时，抽提3小时后，质粒抽提浓度相比于传统方法增加了36-75％；抽提4小时后，质粒抽提浓度相比于传统方法增加了45-95％，尤其是在葡萄糖的浓度为1g/L且阿拉伯糖的浓度为0.6g/L时，抽提4小时后，质粒抽提浓度相比于传统方法增加了95％。
表3实施例1-6与对比实施例2相比质粒抽提浓度百分比
时间 1h 2h 3h 4h 5h
实施例1 16％ -19％ 75％ 45％ 34％
实施例2 14％ -38％ 67％ 95％ 52％
实施例3 2％ -46％ 36％ 63％ 17％
实施例4 70％ -26％ -34％ -7％ -28％
实施例5 47％ -38％ 2％ -11％ -41％
实施例6 5％ -30％ -9％ -12％ -29％
SEQ ID NO：1 pCCIBAC载体序列
Figure PCTCN2019128523-appb-000001
Figure PCTCN2019128523-appb-000002
Figure PCTCN2019128523-appb-000003
Figure PCTCN2019128523-appb-000004
以上对本发明所提供的诱导质粒拷贝数增加的培养基及其应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应 当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (14)

  1. 一种诱导质粒拷贝数增加的培养基，其特征在于，所述培养基包含胰蛋白胨、酵母提取物、葡萄糖、氯化钠和阿拉伯糖。
  2. 根据权利要求1所述的诱导质粒拷贝数增加的培养基，其特征在于，所述葡萄糖的浓度为0.1-10g/L，优选为1-5g/L，更优选为0.5-2g/L。
  3. 根据权利要求1所述的诱导质粒拷贝数增加的培养基，其特征在于，所述阿拉伯糖的浓度为0.1-10g/L，优选为1-5g/L，更优选为0.3-0.75g/L。
  4. 根据权利要求1所述的诱导质粒拷贝数增加的培养基，其特征在于，所述胰蛋白胨的浓度为1-15g/L，优选为5-10g/L。
  5. 根据权利要求1所述的诱导质粒拷贝数增加的培养基，其特征在于，所述酵母提取物的浓度为1-15g/L，优选为5-10g/L。
  6. 根据权利要求1所述的诱导质粒拷贝数增加的培养基，其特征在于，所述氯化钠的浓度为1-15g/L，优选为5-10g/L。
  7. 根据权利要求1-6中任一项所述的诱导质粒拷贝数增加的培养基，其特征在于，所述培养基中还包含氯化镁、氯化钾、氯化铁或氯化钙中的一种或多种。
  8. 根据权利要求1-7中任一项所述的诱导质粒拷贝数增加的培养基，其特征在于，所述质粒为含有oriV复制起始位点的质粒。
  9. 根据权利要求8所述的诱导质粒拷贝数增加的培养基，其特征在于，所述质粒为含有oriV复制起始位点的单拷贝质粒，优选为pCCIBAC质粒。
  10. 一种诱导质粒拷贝数增加的方法，包括以下步骤：
    步骤(1)：将质粒转入大肠杆菌中；
    步骤(2)：将步骤(1)得到的大肠杆菌接种到权利要求1-9中任一项所述的培养基中，在适于培养的条件下进行培养。
  11. 根据权利要求10所述的诱导质粒拷贝数增加的方法，其特征在于，所述步骤(1)中将质粒转入大肠杆菌感受态中。
  12. 根据权利要求10所述的诱导质粒拷贝数增加的方法，其特征在于，所述步骤(1)中的大肠杆菌为EPI300或EPI400大肠杆菌。
  13. 根据权利要求10所述的诱导质粒拷贝数增加的方法，其特征在于，所述步骤(2)中的培养温度为25-37℃，培养时间为4-6h。
  14. 根据权利要求1-9中任一项所述的培养基在诱导质粒拷贝数增加中的应用。
PCT/CN2019/128523 2018-12-27 2019-12-26 诱导质粒拷贝数增加的培养基及其应用 WO2020135549A1 (zh)

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