[自然科学]chapter5-adanced ector.ppt

To minimize the problems associated with high-level expression, it is usual to use a vector the cloned gene is under the control of a regulated promoter. Many different vectors have been constructed for regulated expression of gene inserts most of those contain λPL , T7, trc (tac) or BAD. a family of expression vectors The pET vectors----- utilize phage T7 promoters to regulate synthesis of gene products Strategy for regulating the expression of genes cloned into pET vector compatible 溶菌酶 Constitutive 5.2.3.2 BAC system (bacterial artificial chromosome) base on the single-copy sex factor F of E. coli. Bacterial cloning system for mapping and analysis of complex genomes The oriS and repE genes mediate the unidirectional(单向）replication of the F factor, maintain the copy number at a level of one or two per genome. chloramphenicol（氯霉素）resistance marker cleavage sites for λ terminase cleavage sites P1 cre protein Structure of a BAC vector derived from a mini-F plasmid. unique cleavage sites for excision of inserts. cloning sites λ P1 To generate RNA probes pBAC108L includes cos N and lox P sites, from P1 from λ (SfiI, NotI to facilitate the gene excision) two cloning sites (HindIII and BamHI) several G+C restriction enzyme sites The cloning site is flanked by T7 and SP6 promoters Compositions BAC can be transformed into E. coli very efficiently, and avoiding the packaging extracts be capable of maintaining human and plant genomic fragments of greater than 300 kb for over 100 generations. been used to construct genome libraries with an average insert size of 125 kb required with the P1 system. BACs usages with a high degree of stability feature Two widely used BAC vectors, pBeloBAC11 and pECBAC1, are derivatives of pBAC108L The first BAC vector, pBAC108L, lacked a selectable marker for recombinants. clones with inserts be identified by colony hybridization. defect Two widely used BAC vectors: pBeloBAC11 and pECBAC1, are derivatives of pBAC108L Furthe