[1]严振亚,柴美霞,张　青,等.寡核苷酸介导的重组工程法构建容纳毒性基因ccdB的新型菌株[J].南京师范大学学报(自然科学版),2016,39(02):66.[doi:10.3969/j.issn.1001-4616.2016.02.013]
　Yan Zhenya,Chai Meixia,Zhang Qing,et al.Construction of A New ccdB Tolerant Strain ThroughOligonucleotide Mediated Recombineering[J].Journal of Nanjing Normal University(Natural Science Edition),2016,39(02):66.[doi:10.3969/j.issn.1001-4616.2016.02.013]
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寡核苷酸介导的重组工程法构建容纳毒性基因ccdB的新型菌株()
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《南京师范大学学报》（自然科学版）[ISSN:1001-4616/CN:32-1239/N]

卷:
第39卷
期数:
2016年02期
页码:
66
栏目:
生命科学
出版日期:
2016-06-30

文章信息/Info

Title:
Construction of A New ccdB Tolerant Strain ThroughOligonucleotide Mediated Recombineering
作者:
严振亚; 柴美霞; 张　青; 骆　希; 尚广东
南京师范大学生命科学学院，江苏省微生物工程技术研究中心，江苏省微生物与功能基因组学重点实验室，江苏 南京 210023
Author(s):
Yan Zhenya; Chai Meixia; Zhang Qing; Luo Xi; Shang Guangdong
School of Life Sciences，Nanjing Normal University，Jiangsu Engineering and Technology Research Center for Microbiology，Jiangsu Key Laboratory for Microbes and Functional Genomics， Nanjing 210023，China
关键词:
ccdB; R6K; 寡核苷酸; 重组工程
Keywords:
ccdB; R6K; single-stranded oligonucleotide; recombineering
分类号:
Q819
DOI:
10.3969/j.issn.1001-4616.2016.02.013
文献标志码:
A
摘要:
毒性基因ccdB是最为常用的一种负筛选标记，ccdB基因和R6K复制子一起组成了高效的基因克隆和基因组修饰的遗传操作元件. 为获得高转化效率的容纳ccdB基因的菌株，本研究采用重组工程手段，以经优化去除了错配修复的寡核苷酸与pUC骨架、含有ccdB基因的质粒pMK2010共转化，直接对含pir116基因型的大肠杆菌DH10B衍生菌株的基因组进行修饰. 在筛选的10个菌株中，7个为预期的GyrA462基因型. 为消除pMK2010，构建了一个pUC骨架、氨苄青霉素抗性的诱导自剪切质粒pLS2750. pLS2750通过质粒不相容性去除pMK2010后，经诱导I-SceI自剪切而消除. 所得菌株LS027的电转化效率为6.9×108/mg，是对照菌株的约100倍，R6K质粒呈现高拷贝. 以LS027为宿主菌的构建了系列克隆载体，以只进行基因克隆可避免载体自连的干扰. 新型ccdB基因容纳菌株LS027有着基因操作方面广泛运用的潜力.
Abstract:
Toxic gene ccdB is the most often used counter selection marker，together with R6K replicon，they constitute the highly efficient gene cloning and genome modification genetic elements. To obtain a high transformation efficiency ccdB tolerant strain，in this study，a single-stranded oligonucleotide designed to circumvent the mismatch repair was co-transformed with pUC backbone，ccdB harboring plasmid pMK2010 into Escherichia coli BUN20，a DH10B derivative with pir116 genotype. Seven out of ten strains exhibited the expected GyrA462 genotype. To eliminate pMK2010，a pUC backbone，auto-cleavable vector pLS2750 was constructed. pMK2010 was replaced by pLS2750 under ampicillin selection via incompatibility mechanism，then pLS2750 was self eliminated under the I-SceI self cleavage. The engineered stain LS027 shows an electroporation efficiency of 6.9×108/mg，which is about 100 folder higher than that of the control strain；and R6K plasmid shows high copy number. Serial gene cloning vectors were constructed with LS027 as host，the vector will eliminate self-ligated background. The new ccdB tolerant strain LS027 will find general applications in gene manipulation.

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备注/Memo

备注/Memo:
收稿日期：2015-04-25．　
基金项目：国家自然科学基金（NSFC81273412）.　
通讯联系人：尚广东，副教授，研究方向：合成生物学. E-mail：shanggd@hotmail.com
更新日期/Last Update: 2016-06-30