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The constructed E. coli plasmid pBR322. Features of pBR322 An origin of replication (ori) Two genes that confer resistance to different antibiotics (tet R , amp R ) Several unique recognition sequences (EcoRI, BamH1) Small size (4,361 bp). Cloning a gene into plasmid pBR322.
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The constructed E. coli plasmid pBR322 • Features of pBR322 • An origin of replication (ori) • Two genes that confer resistance to different antibiotics (tetR, ampR) • Several unique recognition sequences (EcoRI, BamH1) • Small size (4,361 bp)
Cloning a gene into plasmid pBR322 • Methods of transformation • → a process to introduce plasmid into cells • Heat shock: 0 to 37 ~ 43 oC in CaCl2 solution • Electrophoration: high voltage
Expression Vector Expression of cloned genes produces large quantities of protein • Components of expression vector • replication origin • polylingker (MRS or MCS) • Selective marker • promoter • operator • ribosome binding site • gene encoding repressor
그림 19.7 외부 DNA 절편은 제한효소를 이용하여 플라스미드 내로 삽입될 수 있다.
pET vector f1 origin Xho I (7218) Hin dIII (6887) kan Nco I (6676) preCGT Hin dIII (5873) Nde I (5851) pET26-preCGT 7379 bp Nde I (5071) lacI
Regulation of Protein Expression in pET System • Double induction by IPTG • T7 RNA polymerase (98 kDa) • target gene (only in T7lac vectors) • Compatible with a wide range of expression hosts • requires DE3 lysogen