募集RNA聚合酶.PPT

第五讲　基因工程的载体 Vectors for Gene Engineering Blue/White Color Screening The E. coli component of a yeast shuttle vector includes an origin of replication and a selectable marker, e.g. antibiotic resistance, Beta lactamase. The yeast component of a yeast shuttle vector includes an autonomously replicating sequence (ARS), a yeast centromere (CEN), and a yeast selectable marker (eg. URA3, a gene that encodes an enzyme for uracil synthesis). 第一个用于基因克隆的天然质粒pSC101，它含有一个EcoRI酶切位点，一个四环素抗性选择记号（Tetr ），插入外源片断后不影响其复制功能，但该质粒分子量较大(9263bp)，拷贝数低。 pSC101 Tcr R 6-3 Ner Sr pSC101 Tcr R 6-3 Ner Sr pSC101 Tcr R 6-3 Ner Sr 转化E. coli Tcr Ner 重组菌落 Stanley N. Cohen科恩 The SC stands for Stanley Cohen 2.3.1 pBR322质粒 pBR322 is a plasmid and for a time was one of the most commonly used E. coli cloning vectors. pBR322 was the first artificial plasmid. Created in 1977, it was named eponymously [ip?nim?s] after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez. 2.3 几种常见的质粒克隆载体 4361bp pBR322 is 4361 base pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene, encoding the ampicillin resistance protein(source plasmid pSE2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101). The plasmid has unique restriction sites for more than forty restriction enzymes. 13 of these 40 sites lie within the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1). 第一部分来源于pSE2124的氨苄青霉素抗性基因（Apr）；第二部分来源于pSC101质粒的四环素抗性基因（Tcr）；第三部分则来源于ColE1的派生质粒PMB1的DNA复制起点（ori） pBR322的第一个优点在于它的大小。作为一个克隆载体，它应该小于10 Kb，否则在提纯的时候很容易导致DNA断裂。pBR322本身只有4361 bp，这就意味着我们很容易纯化它以及由它构建的重组体。即便添加上6 Kb的DNA片段，pBR322的大小仍在可操作的范围内。 pBR322的第二个优点是它有两个抗生素抗性基因。这两个抗药性基因都可以作为该质粒的筛选标志，而且每个抗性基因都拥有几个单酶切位点，这在克隆实验中特别有用。用Pst I, Puv I或Sca I酶切后插入DNA会导致氨苄青霉素抗性基因失活，用BamH I和SalI等限制性内切核酸酶酶切后插入DNA会导致四环素抗性基因失活。有大量的限制性酶切位点可以用作插入失活，这意味着pBR322支持很