Chapter 5 Introduction of DNA into living cells- 副本

1、Chapter 5 Introduction of DNA into living cellsXinjun Yu2015.4.14Contents 5.1 Transformation-the uptake of DNA by bacterial cells 5.2 Identification of recombinants 5.3 Transformation of non-bacterial cellsTwo purposes of cloning To get a large number of recombinant DNA PurificationLigation producti

2、on Unligated vector molecules Unligated DNA fragments Self-ligated vector Recombinant DNA carrying the wrong inserted DNA The desired recombinant DNA molecule5.1 Transformation-the uptake of DNA by bacterial cells Transformation (转化): the introduction of any DNA molecule into any living cell. Compet

3、ent cell(感受态细胞): A culture of bacteria that has been treated to enhance their ability to take up DNA molecules.Reception cell Prokaryotes : E. coli Bacillus subtilis Pseudomonadaceae Streptococcus Actinomycete Eukaryotes : yeast animal insect5.1.1 Not all species of bacteria are equally efficient at

4、 DNA uptake Bacillus and Streptococcus : not easy to be transformed E.coli : physical and chemical treatment to enhance their ability to take up DNA5.1.2 Preparation of competent E.coli cells 50mM CaCl2, 4C : improve DNA binding Heat treatment, 42C: transport DNA into the cytoplasm5.1.3 Selection fo

5、r transformed cells Efficiency of transformation(转化效率): Expressed as transformants per g plasmid DNA. 每微克质粒DNA产生的转化子来表示,如106 107 转化子/g DNA Transformation: low efficient (0.01%), Selectable marker is needed Selective medium: to distinguish transformants from non-transformants Larger DNAs transform le

6、ss efficiency than small DNAs. DNA50kb Fig 5.4 Selecting cells that contain pBR322plasmids by plating onto agar medium containing ampicillin and/or tetracycline.对数生长期大肠杆菌50mmol/L CaCl2 , 4C，30min 加入重组质粒DNA, 冰浴 42C, 2min. 营养培养基 选择性培养基（含抗生素）Fig 5.5 Phenotypic expression. 5.2 Identification of recombin

7、ants Insertional inactivation(插入失活): 把外源DNA片段插入到载体的选择标记基因中而使此基因失活，丧失其原有的表型特征。 To distinguish the recombinant DNA from self-ligated vector.Fig 5.6 Insertional inactivation: (a) The normal, non-recombinant vector molecule carries a gene whose product confers a selectable or identifiable characteristic

8、 on the host cell. (b) This gene is disrupted when new DNA is inserted into the vector; as a result the recombinant host does not display the relevant characteristic.5.2.1 Recombinant selection with pBR322-insertional inactivation of an antibiotic resistance geneFig 5.7 The cloning vector pBR322Fig

9、5.8 Screening for pBR322 recombinants by insertional inactivation of the tetracycline resistance gene.5.2.2 Insertional inactivation does not always involve antibiotic resistance Fig 5.9 The cloning vector pUC818 AmN2H19-galactosidase20Fig 5.10 The rationale behind insertionalInactivation of the lac

10、Z gene carried bypUC8. (a) The bacterial and plasmidgenes complement each other to produceA functional -galactosidase molecule.(b)recombinants are screened by plating onto agar containing x-galAnd IPTG -半乳糖苷酶显色法半乳糖苷酶显色法 (蓝白斑蓝白斑, 互补互补) 载体含有lacZ的蓝白筛选法，将目的序列插入载体含有lacZ基因（如质粒pUC）的多克隆位点，转化大肠杆菌，铺在含氨芐青霉素、IP

11、TG、X-gal的培养基中培养，那么不含重组体的菌落，因合成的-半乳糖苷酶能够降解X-gal生成蓝色的产物，而含重组体的细胞因为lacZ基因被破坏而无法合成-半乳糖苷酶，将保持白色菌斑。这一系统被称为乳糖筛选乳糖筛选 （lac selection）又称蓝白蓝白筛选法或筛选法或 互补互补。5.5 Transformation of non-bacterial cellsDNA precipitated with calcium phosphate (磷酸钙沉淀法 )：动物细胞Liposomes （脂质体）导入法:用人工脂质膜包裹DNA，形成的脂质体（Liposome）可以通过与细胞膜融合而将DN

12、A导入细胞 。 适用于各种细胞Electroporation(电穿孔): a method for increasing DNA uptake by protoplasts through prior exposure to a high voltage, which results in the temporary formation of a small spores in the cell membrane. 适用各种细胞5.5 Transformation of non-bacterial cells Protoplasts transformation（原生质体转化法）： 酵母、真菌

13、和植物细胞 Microinjection(显微注射): a method of introducing new DNA into a cell by injecting it directly into the nucleus. 动植物细胞 Biolistics(生物弹): A means of introducing DNA into cells that involves bombardment with high velocity microprojectiles coated with DNA. 适用于各种细胞Fig 5.14 Strategies for introducing new DNA into animal and plant cellsFig 5.15 Two physical methods for introducing DNA into cellsAssignmentsAssignmentsTransformation; Competent cell; the process of transformation ; Efficiency of transformation; Insertional inactivation; lac selection; E