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Recombinant DNA Technology. Dr. Hui LI Office : S408 Tel: 26538722. Topic 3 Transfer of target gene into receptor cell. Procaryote cells as receptor cells. How to make competent E.coli cells?. Bacteria that are capable of uptaking the foreign DNA are called competent ;
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Recombinant DNA Technology Dr. Hui LI Office : S408 Tel: 26538722
Topic 3 Transfer of target gene into receptor cell
Procaryote cells as receptor cells How to make competent E.coli cells? • Bacteria that are capable of uptaking the foreign DNA are called competent; • To increase the permeability of cell membrane.
Species Use restriction enzymes-deficiency E.coli
Principle Calcium chloride (CaCl2)transformation is a method of promoting competence for bacterial cells.
Procedure Collection of bacterial cells by centrifugation at 4℃ Until OD600 0.3-0.4 E.coli culture On ice 5-10 min Collect cells by centrifugation at 4℃ Suspension of cell pellet with chill CaCl2 On ice 30 min Suspension of cell pellet with chill CaCl2 Suspension of cell pellet with chill CaCl2 Collect cells by centrifugation at 4℃ Store at -70 ℃
How to introduce foreign DNA? Methods of introduction foreign DNA into competent cells （1）转化（transformation） 大肠杆菌捕获质粒DNA的过程。 （2）转染（transfection) 大肠杆菌捕获噬菌体DNA的过程。 （3）转导（transduction) 借助噬菌体把外源DNA导入细菌的过程。
Efficiency Number of colonies / g DNA An excellent preparation of competent cells will give ~108 colonies per microgram of plasmid. Methods of transformation （1） Heat shock(热休克法） 简单，但转化效率不高（106-108/g DNA）。 （2）Electroporation(电转化法) 转化效率高（高于109/g DNA）。
（1）Heat shock Uptake of DNA 10ng vector Adhesion of DNA 42ºC 30s-120s On ice, 10 min 100L competent cells Add 1mL LB culture Incubate at 37ºC for 1h 10-100 L plated on antibiotic selective media (Only transformants can survive)
（2）Electroporation Electroporationis another method of promoting competence. In the method the cells are briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell membrane through which the plasmid DNA enters. This method is amenable to the uptake of large plasmid DNA. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.
Factors affected efficiency of recombination （1）Recombinant vector ①circular plasmid Circular recombinant plasmid Self-ligation of plasmid * Linear plasmids will be degraded once they enter into bacteria
（2）competent cells ①Use the cells at exponential phase ②Prepare under ice condition ③Treat sufficiently with CaCl2 ④Store at -70℃ ⑤Thaw the cells quickly, and never re-store the cells after thawing
Transduction by phages （1）in vitro packaging（体外包装） 把重组的噬菌体DNA或Cosmid质粒DNA包装成具有感染能力的噬菌体颗粒。 （2）Transduction（转导） 通过受体菌细胞表面的DNA接受器位点（receptor site)，使带有外源基因的重组体DNA注入受体大肠杆菌进行扩增。
（3）cI857基因突变的噬菌体 cI857基因是个温度敏感性阻遏蛋白基因，感染细菌后，在32℃下培养细菌时能够保持溶源性。 但当温度升高到44℃—45℃时，就会导致cI基因编码的阻遏蛋白失活，DNA复制、外壳蛋白合成。便于提取外壳蛋白。 但由于该噬菌体的S基因上有一个突变，所以细菌还不裂解。
DNA cI857 其它基因 阻遏 蛋白 溶源状态（DNA不转录、不翻译） 32℃ 阻遏 蛋白 DNA转录、翻译合成外壳蛋白 44℃—45℃
（4） 互补型噬菌体 在cI857基因突变的噬菌体的基础上，选择两种外壳蛋白的突变型噬菌体。 噬菌体1 外壳蛋白基因E发生了无义突变，不能合成头部蛋白，但能合成其它外壳蛋白（尾部蛋白）。 噬菌体2 外壳蛋白基因D发生了无义突变，不能合成头部的包装识别蛋白，但能合成其它外壳蛋白（头部、尾部蛋白）。
（6） 转导 体外包装好的重组噬菌体感染受体菌，使受体菌发生溶菌，形成噬菌斑。（每g DNA能形成106噬菌斑）。
Eukaryote cells as receptor cells I. Yeast cells II. Plant cells III. Animal cells
I. Yeast cells Methods of transformation for yeast cells （1）Protoplast methode Degrade Cell wall (chitinase) CaCl2、 PEG competemt protoplast yeast Vector containing foreign DNA transformation PEG（聚乙二醇）使细胞壁具有通透性，允许DNA进入。 CaCl2使细胞膜具有通透性，允许DNA进入。
（2）Li+ method 0.1mol/L LiCl (Lithium chloride ) competent yeast 40% PEG 4000 Vector containing foreign DNA
II. Plant cells 1. leaf disk mediated by Agrobacterium(叶盘法) 土壤农杆菌浸泡 消毒 切取 叶盘 看护培养基 愈伤组织 分化幼苗
2. Electroporation (电击法) Cellulase or pectinase (纤维素酶和果胶酶) Plant cells protoplast DNA Mix with buffer of electroporation 1-2kV,3-25F 愈伤组织 (Callus) 幼苗 (shoots) make transient holes in cell membranes using electric shock
3. Gene gun（基因枪法） Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. The transformation efficiency is lower than in agribacterial mediated transformation, but most plants can be transformed with this method.
III. Animal cells 1. Calcium phosphate precipitation （1)Principle： HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.
2. Liposome（脂质体） small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells, transfection is better achieved using cationic liposomes (or mixtures), because the cells are more sensitive.
3. Microinjection (显微注射法) Foreign DNA is directly injected into the nucleus of receptor cells
4. DEAE-dextran (DEAE---葡萄糖传染法) 二乙氨乙基（diethyl-aminoehtyl，DEAE）葡萄糖能促进哺乳动物细胞摄入外源DNA。 The negatively charged DNA binds to DEAE and the complex is taken up by the cell via endocytosis. 吸附到细胞表面后被细胞吞入，部分DNA可以进入到细胞核里。
细胞 DEAE-dextran 外源DNA 预处理 摄入 DEAE-dextran 细胞 混合 外源DNA DEAE-dextran is toxic to cells! 5. Via virus
