CN111378609A - 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 PDFInfo
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Abstract
本发明提供了一种诱导质粒拷贝数增加的培养基及其应用,本发明的诱导质粒拷贝数增加的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
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。
技术背景
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。
有益效果
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C6H12O6,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C5H10O5,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.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.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.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.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.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.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.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.对比实施例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9.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。在初始培养基中培养转化的EPI300菌株。初始培养基成分为10g/L胰蛋白胨,5g/L酵母提取物和10g/L氯化钠,2g/L葡萄糖和0.75g/L阿拉伯糖。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。在初始培养基中培养转化的EPI400菌株。初始培养基成分为5g/L胰蛋白胨,10g/L酵母提取物和5g/L氯化钠,1g/L葡萄糖和0.6g/L阿拉伯糖。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。在初始培养基中培养转化的EPI300菌株。初始培养基成分为10g/L胰蛋白胨,10g/L酵母提取物和10g/L氯化钠,0.5g/L葡萄糖和0.3g/L阿拉伯糖。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。在初始培养基中培养转化的EPI400菌株。初始培养基成分为10g/L胰蛋白胨,10g/L酵母提取物和5g/L氯化钠,2g/L葡萄糖和1g/L阿拉伯糖。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。在初始培养基中培养转化的EPI400菌株。初始培养基成分为10g/L胰蛋白胨,10g/L酵母提取物和5g/L氯化钠,10g/L葡萄糖和5g/L阿拉伯糖。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。在初始培养基中培养转化的EPI400菌株。初始培养基成分为10g/L胰蛋白胨,10g/L酵母提取物和5g/L氯化钠,10g/L葡萄糖和2g/L阿拉伯糖。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。在初始培养基中分别培养转化的EPI300菌株。初始培养基成分为10g/L胰蛋白胨,10g/L酵母提取物和10g/L氯化钠。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AxyGENMiniprep 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。在初始培养基中分别培养转化的EPI300菌株。初始培养基成分为10g/L胰蛋白胨,10g/L酵母提取物和10g/L氯化钠。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。质粒抽提结果见图8。
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)
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
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% |
[0001] 序列表
[0002] <110>江苏金斯瑞生物科技有限公司
[0003] <120>诱导质粒拷贝数增加的培养基及其应用
[0004] <160>1
[0005] <170>SIPOSequenceListing 1.0
[0006] <210>1
[0007] <211>8128
[0008] <212>DNA
[0009] <213>人工序列(Artificial Sequence)
[0010] <400>1
[0011] gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg60
[0012] cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccacacagat120
[0013] gcgtaaggag aaaataccgc atcaggcgcc attcgccatt cagctgcgca actgttggga180
[0014] agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc240
[0015] aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc300
[0016] cagtgaattg taatacgact cactataggg cgaattcgag ctcggtaccc ggggatcctc360
[0017] tagagtcgac ctgcaggcat gcaagcttga gtattctata gtctcaccta aatagcttgg420
[0018] cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca480
[0019] acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca540
[0020] cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc600
[0021] attaatgaat cggccaacgc gaaccccttg cggccgcccg ggccgtcgac caattctcat660
[0022] gtttgacagc ttatcatcga atttctgcca ttcatccgct tattatcact tattcaggcg720
[0023] tagcaaccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc780
[0024] cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa840
[0025] acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat900
[0026] ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa960
[0027] ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta1020
[0028] gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac1080
[0029] tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg1140
[0030] aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc1200
[0031] atacgaaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa1260
[0032] aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc1320
[0033] tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat1380
[0034] tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct1440
[0035] cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga1500
[0036] aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt ggcccagggc1560
[0037] ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc ttccgtcaca1620
[0038] ggtatttatt cgcgataagc tcatggagcg gcgtaaccgt cgcacaggaa ggacagagaa1680
[0039] agcgcggatc tgggaagtga cggacagaac ggtcaggacc tggattgggg aggcggttgc1740
[0040] cgccgctgct gctgacggtg tgacgttctc tgttccggtc acaccacata cgttccgcca1800
[0041] ttcctatgcg atgcacatgc tgtatgccgg tataccgctg aaagttctgc aaagcctgat1860
[0042] gggacataag tccatcagtt caacggaagt ctacacgaag gtttttgcgc tggatgtggc1920
[0043] tgcccggcac cgggtgcagt ttgcgatgcc ggagtctgat gcggttgcga tgctgaaaca1980
[0044] attatcctga gaataaatgc cttggccttt atatggaaat gtggaactga gtggatatgc2040
[0045] tgtttttgtc tgttaaacag agaagctggc tgttatccac tgagaagcga acgaaacagt2100
[0046] cgggaaaatc tcccattatc gtagagatcc gcattattaa tctcaggagc ctgtgtagcg2160
[0047] tttataggaa gtagtgttct gtcatgatgc ctgcaagcgg taacgaaaac gatttgaata2220
[0048] tgccttcagg aacaatagaa atcttcgtgc ggtgttacgt tgaagtggag cggattatgt2280
[0049] cagcaatgga cagaacaacc taatgaacac agaaccatga tgtggtctgt ccttttacag2340
[0050] ccagtagtgc tcgccgcagt cgagcgacag ggcgaagccc tcgagctggt tgccctcgcc2400
[0051] gctgggctgg cggccgtcta tggccctgca aacgcgccag aaacgccgtc gaagccgtgt2460
[0052] gcgagacacc gcggccggcc gccggcgttg tggatacctc gcggaaaact tggccctcac2520
[0053] tgacagatga ggggcggacg ttgacacttg aggggccgac tcacccggcg cggcgttgac2580
[0054] agatgagggg caggctcgat ttcggccggc gacgtggagc tggccagcct cgcaaatcgg2640
[0055] cgaaaacgcc tgattttacg cgagtttccc acagatgatg tggacaagcc tggggataag2700
[0056] tgccctgcgg tattgacact tgaggggcgc gactactgac agatgagggg cgcgatcctt2760
[0057] gacacttgag gggcagagtg ctgacagatg aggggcgcac ctattgacat ttgaggggct2820
[0058] gtccacaggc agaaaatcca gcatttgcaa gggtttccgc ccgtttttcg gccaccgcta2880
[0059] acctgtcttt taacctgctt ttaaaccaat atttataaac cttgttttta accagggctg2940
[0060] cgccctgtgc gcgtgaccgc gcacgccgaa ggggggtgcc cccccttctc gaaccctccc3000
[0061] ggtcgagtga gcgaggaagc accagggaac agcacttata tattctgctt acacacgatg3060
[0062] cctgaaaaaa cttcccttgg ggttatccac ttatccacgg ggatattttt ataattattt3120
[0063] tttttatagt ttttagatct tcttttttag agcgccttgt aggcctttat ccatgctggt3180
[0064] tctagagaag gtgttgtgac aaattgccct ttcagtgtga caaatcaccc tcaaatgaca3240
[0065] gtcctgtctg tgacaaattg cccttaaccc tgtgacaaat tgccctcaga agaagctgtt3300
[0066] ttttcacaaa gttatccctg cttattgact cttttttatt tagtgtgaca atctaaaaac3360
[0067] ttgtcacact tcacatggat ctgtcatggc ggaaacagcg gttatcaatc acaagaaacg3420
[0068] taaaaatagc ccgcgaatcg tccagtcaaa cgacctcact gaggcggcat atagtctctc3480
[0069] ccgggatcaa aaacgtatgc tgtatctgtt cgttgaccag atcagaaaat ctgatggcac3540
[0070] cctacaggaa catgacggta tctgcgagat ccatgttgct aaatatgctg aaatattcgg3600
[0071] attgacctct gcggaagcca gtaaggatat acggcaggca ttgaagagtt tcgcggggaa3660
[0072] ggaagtggtt ttttatcgcc ctgaagagga tgccggcgat gaaaaaggct atgaatcttt3720
[0073] tccttggttt atcaaacgtg cgcacagtcc atccagaggg ctttacagtg tacatatcaa3780
[0074] cccatatctc attcccttct ttatcgggtt acagaaccgg tttacgcagt ttcggcttag3840
[0075] tgaaacaaaa gaaatcacca atccgtatgc catgcgttta tacgaatccc tgtgtcagta3900
[0076] tcgtaagccg gatggctcag gcatcgtctc tctgaaaatc gactggatca tagagcgtta3960
[0077] ccagctgcct caaagttacc agcgtatgcc tgacttccgc cgccgcttcc tgcaggtctg4020
[0078] tgttaatgag atcaacagca gaactccaat gcgcctctca tacattgaga aaaagaaagg4080
[0079] ccgccagacg actcatatcg tattttcctt ccgcgatatc acttccatga cgacaggata4140
[0080] gtctgagggt tatctgtcac agatttgagg gtggttcgtc acatttgttc tgacctactg4200
[0081] agggtaattt gtcacagttt tgctgtttcc ttcagcctgc atggattttc tcatactttt4260
[0082] tgaactgtaa tttttaagga agccaaattt gagggcagtt tgtcacagtt gatttccttc4320
[0083] tctttccctt cgtcatgtga cctgatatcg ggggttagtt cgtcatcatt gatgagggtt4380
[0084] gattatcaca gtttattact ctgaattggc tatccgcgtg tgtacctcta cctggagttt4440
[0085] ttcccacggt ggatatttct tcttgcgctg agcgtaagag ctatctgaca gaacagttct4500
[0086] tctttgcttc ctcgccagtt cgctcgctat gctcggttac acggctgcgg cgagcgctag4560
[0087] tgataataag tgactgaggt atgtgctctt cttatctcct tttgtagtgt tgctcttatt4620
[0088] ttaaacaact ttgcggtttt ttgatgactt tgcgattttg ttgttgcttt gcagtaaatt4680
[0089] gcaagattta ataaaaaaac gcaaagcaat gattaaagga tgttcagaat gaaactcatg4740
[0090] gaaacactta accagtgcat aaacgctggt catgaaatga cgaaggctat cgccattgca4800
[0091] cagtttaatg atgacagccc ggaagcgagg aaaataaccc ggcgctggag aataggtgaa4860
[0092] gcagcggatt tagttggggt ttcttctcag gctatcagag atgccgagaa agcagggcga4920
[0093] ctaccgcacc cggatatgga aattcgagga cgggttgagc aacgtgttgg ttatacaatt4980
[0094] gaacaaatta atcatatgcg tgatgtgttt ggtacgcgat tgcgacgtgc tgaagacgta5040
[0095] tttccaccgg tgatcggggt tgctgcccat aaaggtggcg tttacaaaac ctcagtttct5100
[0096] gttcatcttg ctcaggatct ggctctgaag gggctacgtg ttttgctcgt ggaaggtaac5160
[0097] gacccccagg gaacagcctc aatgtatcac ggatgggtac cagatcttca tattcatgca5220
[0098] gaagacactc tcctgccttt ctatcttggg gaaaaggacg atgtcactta tgcaataaag5280
[0099] cccacttgct ggccggggct tgacattatt ccttcctgtc tggctctgca ccgtattgaa5340
[0100] actgagttaa tgggcaaatt tgatgaaggt aaactgccca ccgatccaca cctgatgctc5400
[0101] cgactggcca ttgaaactgt tgctcatgac tatgatgtca tagttattga cagcgcgcct5460
[0102] aacctgggta tcggcacgat taatgtcgta tgtgctgctg atgtgctgat tgttcccacg5520
[0103] cctgctgagt tgtttgacta cacctccgca ctgcagtttt tcgatatgct tcgtgatctg5580
[0104] ctcaagaacg ttgatcttaa agggttcgag cctgatgtac gtattttgct taccaaatac5640
[0105] agcaatagca atggctctca gtccccgtgg atggaggagc aaattcggga tgcctgggga5700
[0106] agcatggttc taaaaaatgt tgtacgtgaa acggatgaag ttggtaaagg tcagatccgg5760
[0107] atgagaactg tttttgaaca ggccattgat caacgctctt caactggtgc ctggagaaat5820
[0108] gctctttcta tttgggaacc tgtctgcaat gaaattttcg atcgtctgat taaaccacgc5880
[0109] tgggagatta gataatgaag cgtgcgcctg ttattccaaa acatacgctc aatactcaac5940
[0110] cggttgaaga tacttcgtta tcgacaccag ctgccccgat ggtggattcg ttaattgcgc6000
[0111] gcgtaggagt aatggctcgc ggtaatgcca ttactttgcc tgtatgtggt cgggatgtga6060
[0112] agtttactct tgaagtgctc cggggtgata gtgttgagaa gacctctcgg gtatggtcag6120
[0113] gtaatgaacg tgaccaggag ctgcttactg aggacgcact ggatgatctc atcccttctt6180
[0114] ttctactgac tggtcaacag acaccggcgt tcggtcgaag agtatctggt gtcatagaaa6240
[0115] ttgccgatgg gagtcgccgt cgtaaagctg ctgcacttac cgaaagtgat tatcgtgttc6300
[0116] tggttggcga gctggatgat gagcagatgg ctgcattatc cagattgggt aacgattatc6360
[0117] gcccaacaag tgcttatgaa cgtggtcagc gttatgcaag ccgattgcag aatgaatttg6420
[0118] ctggaaatat ttctgcgctg gctgatgcgg aaaatatttc acgtaagatt attacccgct6480
[0119] gtatcaacac cgccaaattg cctaaatcag ttgttgctct tttttctcac cccggtgaac6540
[0120] tatctgcccg gtcaggtgat gcacttcaaa aagcctttac agataaagag gaattactta6600
[0121] agcagcaggc atctaacctt catgagcaga aaaaagctgg ggtgatattt gaagctgaag6660
[0122] aagttatcac tcttttaact tctgtgctta aaacgtcatc tgcatcaaga actagtttaa6720
[0123] gctcacgaca tcagtttgct cctggagcga cagtattgta taagggcgat aaaatggtgc6780
[0124] ttaacctgga caggtctcgt gttccaactg agtgtataga gaaaattgag gccattctta6840
[0125] aggaacttga aaagccagca ccctgatgcg accacgtttt agtctacgtt tatctgtctt6900
[0126] tacttaatgt cctttgttac aggccagaaa gcataactgg cctgaatatt ctctctgggc6960
[0127] ccactgttcc acttgtatcg tcggtctgat aatcagactg ggaccacggt cccactcgta7020
[0128] tcgtcggtct gattattagt ctgggaccac ggtcccactc gtatcgtcgg tctgattatt7080
[0129] agtctgggac cacggtccca ctcgtatcgt cggtctgata atcagactgg gaccacggtc7140
[0130] ccactcgtat cgtcggtctg attattagtc tgggaccatg gtcccactcg tatcgtcggt7200
[0131] ctgattatta gtctgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctgga7260
[0132] accacggtcc cactcgtatc gtcggtctga ttattagtct gggaccacgg tcccactcgt7320
[0133] atcgtcggtc tgattattag tctgggacca cgatcccact cgtgttgtcg gtctgattat7380
[0134] cggtctggga ccacggtccc acttgtattg tcgatcagac tatcagcgtg agactacgat7440
[0135] tccatcaatg cctgtcaagg gcaagtattg acatgtcgtc gtaacctgta gaacggagta7500
[0136] acctcggtgt gcggttgtat gcctgctgtg gattgctgct gtgtcctgct tatccacaac7560
[0137] attttgcgca cggttatgtg gacaaaatac ctggttaccc aggccgtgcc ggcacgttaa7620
[0138] ccgggctgca tccgatgcaa gtgtgtcgct gtcgacgagc tcgcgagctc ggacatgagg7680
[0139] ttgccccgta ttcagtgtcg ctgatttgta ttgtctgaag ttgtttttac gttaagttga7740
[0140] tgcagatcaa ttaatacgat acctgcgtca taattgatta tttgacgtgg tttgatggcc7800
[0141] tccacgcacg ttgtgatatg tagatgataa tcattatcac tttacgggtc ctttccggtg7860
[0142] atccgacagg ttacggggcg gcgacctcgc gggttttcgc tatttatgaa aattttccgg7920
[0143] tttaaggcgt ttccgttctt cttcgtcata acttaatgtt tttatttaaa ataccctctg7980
[0144] aaaagaaagg aaacgacagg tgctgaaagc gagctttttg gcctctgtcg tttcctttct8040
[0145] ctgtttttgt ccgtggaatg aacaatggaa gtccgagctc atcgctaata acttcgtata8100
[0146] gcatacatta tacgaagtta tattcgat 8128
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。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1193354A (zh) * | 1996-03-13 | 1998-09-16 | 宝酒造株式会社 | 质粒 |
CN103205391A (zh) * | 2013-04-12 | 2013-07-17 | 浙江大学 | 一种基因工程菌及其应用 |
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CN104651263A (zh) * | 2014-12-22 | 2015-05-27 | 广西大学 | 一种提高质粒dna含量在大肠杆菌中菌比含量的培养基 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1193354A (zh) * | 1996-03-13 | 1998-09-16 | 宝酒造株式会社 | 质粒 |
CN103205391A (zh) * | 2013-04-12 | 2013-07-17 | 浙江大学 | 一种基因工程菌及其应用 |
Non-Patent Citations (4)
Title |
---|
JADWIGA WILD ET AL.,: ""Copy-Control pBAC/oriV Vectors for Genomic Cloning"", 《METHODS MOL BIOL》 * |
JADWIGA WILD ET AL.,: ""Copy-Control Tightly Regulated Expression Vectors Based on pBAC/oriV"", 《METHODS MOL BIOL》 * |
张海艳: ""植物合成生物学工具箱的优化及其在基因组编辑中的应用研究"", 《中国博士学位论文全文数据库基础科学辑》 * |
郭舒扬: ""拟无枝菌酸菌CP2808中Ansacarbamitocins的生物合成"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
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