2.质粒载体相关知识介绍 The selectable marker usually used is an antibiotic resistance gene This means that bacterial cells carrying the plasmid will be resistant to an antibiotic incorporated into the growth medium. Non-plasmid bearing cells will be killed off. Common antibiotic resistance markers include ampicillin, tetracycline, chloramphenicol and kanamycin. In order to insert a fragment of DNa into a plasmid, it must contain a recognition site for a restriction endonuclease To be useful, the site must only occur once in the vector sequence. In most commercial vectors, this is achieved by use of a polylinker. This is a length of dNa which is engineered to have a series of restriction sites next to each other
The selectable marker usually used is an antibiotic resistance gene. This means that bacterial cells carrying the plasmid will be resistant to an antibiotic incorporated into the growth medium. Non-plasmid bearing cells will be killed off. Common antibiotic resistance markers include ampicillin, tetracycline, chloramphenicol and kanamycin. In order to insert a fragment of DNA into a plasmid, it must contain a recognition site for a restriction endonuclease. To be useful, the site must only occur once in the vector sequence. In most commercial vectors, this is achieved by use of a polylinker. This is a length of DNA which is engineered to have a series of restriction sites next to each other. 2. 质粒载体相关知识介绍
2.质粒载体相关知识介绍 (1)α-互补( alpha- complementation 大肠杄菌β-半乳糖苷酶可以和其底物XGa相互作 用并且释放出一种蓝色物质,当该酶的a-片段和-片 段分开时就失去了这种显色的功能。通常将编码该酶 α-片段的LcZ基因插入到载体的多克隆位点的侧翼序 列中,而在一些人工构建的大肠杆菌株系中却只能编 码产生该酶的o片段。这样一来,含有功能性完整的 LacZ基因的载体导入到这类寄主细胞中时,载体编码 的α-片段就能和寄主编码的o片段发生互补并具有了 对底物Xgal的作用功能(发生显色反应),这种现象 被成为是wpha- complementation
（1） -互补 (alpha-complementation) 大肠杆菌-半乳糖苷酶 可以和其底物X-Gal 相互作 用并且释放出一种蓝色物质，当该酶的-片段和-片 段分开时就失去了这种显色的功能。通常将编码该酶 -片段的LacZ 基因插入到载体的多克隆位点的侧翼序 列中，而在一些人工构建的大肠杆菌株系中却只能编 码产生该酶的片段。这样一来，含有功能性完整的 LacZ 基因的载体导入到这类寄主细胞中时，载体编码 的 -片段就能和寄主编码的片段发生互补并具有了 对底物X-gal的作用功能（发生显色反应），这种现象 被成为是 alpha-complementation. 2. 质粒载体相关知识介绍
2.质粒载体相关知识介绍 (1)wpha- complementation(0-互补) TheE. coli beta-galactosidase(β-半乳糖苷酶)can interact with substrate X-Gal and release blue substance. The Lac gene encodes the alpha fragment(α片段) of the enzyme was flan ked by the polylinker in some vectors and some host strain of E. coli produce the omega fragment( H E)of the enzyme Transformation of the host with these vectors which coded alpha fragment, then allows production of a functional beta-galactosidase, a phenomenon known as alpha-complementation
（1）alpha-complementation(-互补) The E. coli beta-galactosidase(-半乳糖苷酶) can interact with substrate X-Gal and release blue substance. The LacZ gene encodes the alpha fragment(片段) of the enzyme was flanked by the polylinker in some vectors and some host strain of E. coli produce the omega fragment(片段)of the enzyme. Transformation of the host with these vectors, which coded alpha fragment, then allows production of a functional beta-galactosidase, a phenomenon known as alpha-complementation. 2. 质粒载体相关知识介绍
2.质粒载体相关知识介绍 所以含有这类载体的菌落就很容易在含有底物X Gal和诱导物ITG的平板上分辨出来。因为这类菌落中 释放出的蓝 E. col 色物质可以 Vector 将整个菌落 Lacz 染成蓝色, Functional B-galactosidase 非常容易辨m zyme 别 B-galactosidase A-fragment Blue colonies on X-Gal/PTG plates Chromosome
所以含有这类载体的菌落就很容易在含有底物XGal和诱导物IPTG的平板上分辨出来。因为这类菌落中 释放出的 蓝 色物质可以 将整个菌落 染成蓝色 ， 非常容易辨 别。 2. 质粒载体相关知识介绍
2.质粒载体相关知识介绍 Colonies containing plasmid can then be identified by growth on plates containing X-Gal and an inducer of the enzyme E. col IPTG Vector Plasmid Lacz containing Functional colonies B-galactosidase zyme a-fragment release the B-galactosidase chromophore A-fragment Blue colonies on X-Gal/PTG plates from the X Gal and are Chromosome coloured blue
Colonies containing plasmid can then be identified by growth on plates containing X-Gal and an inducer of the enzyme IPTG, Plasmid containing colonies release the chromophore from the XGal and are coloured blue. 2. 质粒载体相关知识介绍