1 / 7

The constructed E. coli plasmid pBR322

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.

isaiah
Download Presentation

The constructed E. coli plasmid pBR322

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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)

  2. 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

  3. Identification of cell containing plasmid with target gene

  4. 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

  5. 그림 19.7 외부 DNA 절편은 제한효소를 이용하여 플라스미드 내로 삽입될 수 있다.

  6. 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

  7. 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

More Related