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 (轉(zhuǎn)化): the introduction of any DNA molecule into any living cell. Compet

3、ent cell(感受態(tài)細(xì)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(轉(zhuǎn)化效率): Expressed as transformants per g plasmid DNA. 每微克質(zhì)粒DNA產(chǎn)生的轉(zhuǎn)化子來表示,如106 107 轉(zhuǎn)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.對(duì)數(shù)生長期大腸桿菌50mmol/L CaCl2 , 4C，30min 加入重組質(zhì)粒DNA, 冰浴 42C, 2min. 營養(yǎng)培養(yǎng)基 選擇性培養(yǎng)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片段插入到載體的選擇標(biāo)記基因中而使此基因失活，喪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 -半乳糖苷酶顯色法半乳糖苷酶顯色法 (藍(lán)白斑藍(lán)白斑, 互補(bǔ)互補(bǔ)) 載體含有l(wèi)acZ的藍(lán)白篩選法，將目的序列插入載體含有l(wèi)acZ基因（如質(zhì)粒pUC）的多克隆位點(diǎn)，轉(zhuǎn)化大腸桿菌，鋪在含氨芐青霉素、IP

11、TG、X-gal的培養(yǎng)基中培養(yǎng)，那么不含重組體的菌落，因合成的-半乳糖苷酶能夠降解X-gal生成藍(lán)色的產(chǎn)物，而含重組體的細(xì)胞因?yàn)閘acZ基因被破壞而無法合成-半乳糖苷酶，將保持白色菌斑。這一系統(tǒng)被稱為乳糖篩選乳糖篩選 （lac selection）又稱藍(lán)白藍(lán)白篩選法或篩選法或 互補(bǔ)互補(bǔ)。5.5 Transformation of non-bacterial cellsDNA precipitated with calcium phosphate (磷酸鈣沉淀法 )：動(dòng)物細(xì)胞Liposomes （脂質(zhì)體）導(dǎo)入法:用人工脂質(zhì)膜包裹DNA，形成的脂質(zhì)體（Liposome）可以通過與細(xì)胞膜融合而將DN

12、A導(dǎo)入細(xì)胞 。 適用于各種細(xì)胞Electroporation(電穿孔): 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. 適用各種細(xì)胞5.5 Transformation of non-bacterial cells Protoplasts transformation（原生質(zhì)體轉(zhuǎn)化法）： 酵母、真菌

13、和植物細(xì)胞 Microinjection(顯微注射): a method of introducing new DNA into a cell by injecting it directly into the nucleus. 動(dòng)植物細(xì)胞 Biolistics(生物彈): A means of introducing DNA into cells that involves bombardment with high velocity microprojectiles coated with DNA. 適用于各種細(xì)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