PPT《H Cloning vectors》

100 restriction site , and scores of single restriction site for each restriction enzyme.It and its derivatives contain two antibiotic resistance genes, ampr and tetA. If a target DNA fragment is ligated into the coding region of one of the resistance genes, say tetA, the gene will become insertionally inactivated, and the presence or otherwise of a recombinant product can be determined by the antibiotic resistance exhibited by the transformants. Replica plating Inclusion of tetracycline in the original selection plate will kill off the recombinant colonies in which we are interested.The colonies grown on a normal ampicillin plate are transferred, using an abosorbent pad, to a second plate containing tetracycline.Colonies which grown on this plate probably contain vector plasmids whereas those which do not are likely to be recombinants. The latter may be picked from the ampicillin plate for further analysis. Blue-white screening A more sophisticated procedure for screening for the presence of recombinant plasmids.Only on a single transformation plateThis method also involves the insertional inactivation of a gene and uses the production of a blue compound as an indicator.The gene in this case is lacZ, which encodes the enzyme βCgalactosidase, and is under the control of the lac promoter. If the host E.coli strain is expressing the lac repressor, 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. Insetional inactivation of lacZ in the production of a recombinant plasmid would prevent the development of the blue color.The transformed cells are spread on to a plate containing ampicillin, IPTG and X-gal, to yield a mixture of blue and white colonies.The white colonies have no expressed β-galactosidase and are hence likely to contain the inserted target fragment. The blue colonies probably contain religated vector. pUC质粒 pUC质粒系列也是在pBR322基础上改建成的.pUC的结构: 它含有pBR322的大部分,包括完整的ampr基因和复制起始点.去除了pBR322的tetr区段,换用了M13噬菌体的476bp片段,含LacZ 基因及其启动子的操纵基因、M13的多聚接头polylinker.三大优点 三大优点 分子量更小,仅为2.7KB,容纳外源DNA量增大;

具有更高的拷贝数(每个细胞含500-700个拷贝).含易于检测是否有外源DNA插入的标记基因LacZα,可利用?-互补原理进行蓝白筛选.多克隆位点区(the multiple cloning site, MCS)由人工合成的多个单一酶切位点构成. 其MCS区与M13mp噬菌体载体的相同,可使pUC上克隆的目的基因直接转移到M13mp载体,进行DNA测序和体外突变等研究. pUC18 / 19: 重要的大肠杆菌质粒载体拷贝数

2000 -

3000 / cell装有多克隆位点(MCS)正选择颜色标记 lacZ'

用于基因克隆和测序 质粒载体缺点 ? CaCl2转化效率低? 克隆片段小于10kb? 大量筛选时需要细胞裂解 H2 bacteriophage vector Characteristics of bacteriophageBacteriophage λM13Virus 噬菌体和病毒作为载体的优势 噬菌体或病毒是一类非细胞微生物,能高效率高特异性地侵染宿主细胞,然后或自主复制繁殖,或整合入宿主基因组中潜伏起来,而且在一定的条件下上述两种状态还会相互转化.噬菌体或病毒的上述特性使得它们的DNA能被开发成为基因工程的有用载体,因为: ? 高效率的感染性能使外源基因高效导入受体细胞 ? 自主复制繁殖性能使外源基因在受体细胞中高效扩增 Bacteriophage λ Bacteriophage λ, which infects E.coli cells, can be used as a cloning vector. The process of infection by phage λ Structure of phage λλ-DNA载体的构建λ-DNA载体的主要类型λ-DNA重组分子的体外包装λ-DNA及其重组分子的分离纯化λ-DNA作为载体的优点 λ-噬菌体的生物结构 大肠杆菌的温和型噬菌体由外壳包装蛋白和λ-DNA组成 线性dsDNA病毒,具有末端互补的12nt(cos),感染细菌后粘端互补成双链环状全长48502个核苷酸λ-DNA上至少有50多个基因 λ噬菌体DNA的4个分区 结构区:A~ J共19个基因,长约20kb,其中的基因编码构成头部、尾部、尾丝对组装完整噬菌体所需要的蛋白质重组区 :att、int及xis,长约20kb,是λDNA整合和切出,溶原生长所需的序列调控区:长约10kb,包括启动子、终止子和N、CI基因.控制溶菌和溶原生长最重要的调控基因和序列、以及λDNA复制均在该区域内O-R 为裂解区 λ-DNA载体的构建 缩短长度删除并和增加酶切位点加装选择标记构建琥珀密码子的突变体 缩短长度 野生型λ-DNA包装的上限为51kb,本身长度为48.5kb,只有当插入的外源DNA片段不大于2.5kb时,才能被包装成有感染力的噬菌体颗粒.因此缩短野生型λ-DNA的长度,可以提高装载量.野生型λ-DNA上约有40-50%的片段是复制和裂解所非必需的.根据切除片段长度的不同,可将λ-DNA分成两大类载体:插入型载体取代型载体 删除和增加酶切位点 删除重复的酶切位点 野生型的λ-DNA链上有5个EcoRI 位点和7个HindIII位点,不利于重组操作,必须删除至1 - 2个.增加新的单一酶切位点 为了便于各种来源的DNA片段的克隆,还需........