蓝白斑筛选ppt课件
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LacZ':半乳糖苷酶 IPTG:异丙基-β-d-硫代半乳糖苷 X-gal: 半乳糖苷
2
(启动Fra Baidu bibliotek)
(多克隆位点)
(氨比西林 耐药性基因)
(为蓝白斑筛选设计的质粒载体) 备注:野生型大肠杆菌产生的β -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 [sə'fɪstɪkeɪtɪd] 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
8
• These vectors must be transformed into a special host strain which contains a mutant ['mjuːt(ə)nt] gene expressing only the c-terminal portion of β-galactosidase which can then complement the α-peptide['peptaɪd] to produce active enzyme. This reduces the size of the plasmid-borne gene, but does not alter the basis of the method. • 这些载体必须被转换到特殊的受体菌,它们含有 只表达β半乳糖苷酶部分C段的突变基因,能补充 α肽产生活性酶。这减少了质粒承载基因的大小, 但不能改变这种方法的根本。
• 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.
5
• If the host E. coli Strain is expressing the lac repressor [rɪ'prɛsɚ], 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.
蓝白斑筛选
蓝白斑筛选简述
•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。
• In practice,the vectors used in this method have a shortened derivative of LacZ', which produces the N-terminal,α-peptide of βgalactosidase. • 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β半乳糖苷酶的α肽。
• 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 βgalactosidase 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β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。
• 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 β-galactosidase [ɡə'læktosɪ,des], and is under the control of the lac promoter . • 在这里的基因叫LacZ' ,是编码β半乳糖苷酶,以 及是在lac启动子控制下的。
2
(启动Fra Baidu bibliotek)
(多克隆位点)
(氨比西林 耐药性基因)
(为蓝白斑筛选设计的质粒载体) 备注:野生型大肠杆菌产生的β -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 [sə'fɪstɪkeɪtɪd] 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
8
• These vectors must be transformed into a special host strain which contains a mutant ['mjuːt(ə)nt] gene expressing only the c-terminal portion of β-galactosidase which can then complement the α-peptide['peptaɪd] to produce active enzyme. This reduces the size of the plasmid-borne gene, but does not alter the basis of the method. • 这些载体必须被转换到特殊的受体菌,它们含有 只表达β半乳糖苷酶部分C段的突变基因,能补充 α肽产生活性酶。这减少了质粒承载基因的大小, 但不能改变这种方法的根本。
• 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.
5
• If the host E. coli Strain is expressing the lac repressor [rɪ'prɛsɚ], 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.
蓝白斑筛选
蓝白斑筛选简述
•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。
• In practice,the vectors used in this method have a shortened derivative of LacZ', which produces the N-terminal,α-peptide of βgalactosidase. • 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β半乳糖苷酶的α肽。
• 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 βgalactosidase 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β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。
• 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 β-galactosidase [ɡə'læktosɪ,des], and is under the control of the lac promoter . • 在这里的基因叫LacZ' ,是编码β半乳糖苷酶,以 及是在lac启动子控制下的。