蓝白斑筛选课件

Blue-white screening 蓝白斑筛选,生技1132班 24号颜淑影,1,Blue-white screening: Insertional inactivation of the LacZ gene on a plasmid can be used to screen for recombinants on a plate containing IPTG and X-gal. The X-gal is converted to a blue product if the LacZ gene is intact and induced by IPTG; hence recombinants grow as white colonies. 蓝白斑的筛选：在一个质粒上LacZ 基因的插入失活能够在含有IPTG和X-gal的平板上用来筛选重组株。如果LacZ 基因完好无缺并且通过IPTG诱导，那么X-gal最终将被改变成一种蓝色产物，因此重组株以白色菌落生长。 LacZ：半乳糖苷酶 IPTG：异丙基-d-硫代半乳糖苷 X-gal: 半乳糖苷,蓝白斑筛选简述,2,（多克隆位点）,（为蓝白斑筛选设计的质粒载体）,（启动子）,（氨比西林耐药性基因）,备注：野生型大肠杆菌产生的-半乳糖苷酶可以将无色化合物X-gal切割成半乳糖和深蓝色的物质。,3,蓝白斑筛选具体内容,A more sophisticated sfstketd procedure for screening for the presence of recombinant plasmids, which can be carried out on a single transformation plate, is called blue-white screening. 一个更加复杂的筛选重组株质粒存在的程序，能被利用到单一的转换平板上，被称为蓝白斑筛选。,4,This method also involves the insertional inactivation of a gene and, as the name implies, uses the production of a blue compound as an indicator. 这种方法也涉及到基因的插入灭活，顾名思义，利用一种蓝色复合物的产物作为一种指示剂。 The gene in this case is LacZ , which encodes the enzyme -galactosidase lktos,des, and is under the control of the lac promoter . 在这里的基因叫LacZ ，是编码半乳糖苷酶，以及是在lac启动子控制下的。,5,If the host E. coli Strain is expressing the lac repressor rprs, then expression of a LacZ gene on the vector may be induced using IPTG , and the expressed enzyme can utilize the synthetic substrate X-gal to yield a blue product. 如果宿主大肠杆菌株是正在表达lac 阻遏蛋白，然后在载体上一个LacZ 基因的表达可以诱导利用IPTG，接着表达的酶可以利用人工合成的X-gal培养基产出一种蓝色的产物。,6,In this method (Fig. 2), the transformed cells are spread on to a plate containing ampicillin (to select for transformants in the usual way), IPTG and X-gal, to yield a mixture of blue and white colonies.Insertional inactivation of LacZ in the production of a recombinant plasmid would prevent the development of the blue color. 在这种方法中，转换细胞被传播在一个人含有氨比西林（用正常的方法选择转换株）、IPTG 和 X-gal的平板上产出蓝色和白色菌落的混合物。重组质粒中LacZ基因的产物的插入失活将有碍蓝色物质的形成。,7,The white colonies have no expressed -galactosidase and are hence likely to contain the inserted target fragment. The blue colonies probably contain religated vector. 白色菌落没有表达半乳糖苷酶，因此可能含有插入的目标片段。蓝色菌落可能含有重组载体。 In practice，the vectors used in this method have a shortened derivative of LacZ, which produces the N-terminal，-peptide of -galactosidase. 事实上，利用这种方法的载体含有一种LacZ缩短的衍生物，它生产N末端的半乳糖苷酶的肽。,8,These vectors must be transformed into a special host strain which contains a mutant mjut()nt gene expressing only the c-terminal portion of -galactosidase which can then complement the -peptidepeptad to produce active enzyme. This reduces the size of the plasmid-borne gene,