[]基因工程.ppt

Mutagens Isolation of Mutants Pigmented and nonpigmented mutants Antibiotic-resistant mutants within the inhibition zone Genetic marker, such as the expression of b-galactosidase that produces the blue color Replica Plating Technique: Isolation of Mutants Replica Plating Technique: Isolation of Mutants Penicillin-Selection Method Penicillin kills only growing cells. If penicillin is added to a population growing in a medium lacking the growth factor required by the designed mutant, the parent cells will be killed, whereas the non-growing mutant cells will be unaffected. Mutants that are detectable Non-motile Non-capsulated Rough colony Nutritional Sugar fermentation Drug resistant Virus resistant Temperature sensitive Pigmentless Cold sensitive Molecular basis of Mutation Point Mutation Missense mutation Nonsense mutation Silent mutation Frameshift mutations Occur in protein-encoding gene that including the reading frame If occurs in promoter region, it is not frameshift Back Mutations or Reversions Point Mutations are reversible Revertant wild type phenotype that was lost in the mutant is restored. Same site revertants True revertants Second site mutation Suppressor mutation: restore the wild type phenotype by mutation somewhere else. Mutations involving many base pairs Deletions: mutations in which a region of the DNA has been eliminated Insertions: occur when new bases are added to the DNA, can be a single base or many bases (called insertion sequences) Translocation: large section of chromosomal DNA is moved to a new location. Rates of Mutation Spontaneous mutation: 10-6, Transposition: 10-4 Nonsense mutation: 10-6-10-8 Missense mutation: 10-6-10-8 RNA分子中尿嘧啶(Uracil,U)代替胸腺嘧啶(T) 用噬菌体载体，表达特定基因的蛋白，然后用免疫方法检测 Overlay nitrocellulose fibre 一般说来（除个别病毒）从细菌到哺乳动物的所有生物有机体，它们的基因都是有都是由DNA构成的。由于所有生物的DNA结构都是一致的。因此，来自不同生命形态的基因(DNA)都可以融为一体。由此可见，基因的DNA共性，使进行基因工程的主要理论之一 安全的基因工程载体：要求载体失去自我迁移能力 要求受体细胞安全：如大肠杆菌K12，”安全“特征之一是，是一种营养突变型，必须再有二氨基庚二酸(Diaminopimelic acid, DAP)和胸腺嘧啶核苷酸的培养基中才能生