Agrobacterium Mediated Gene Transfer

Nature’s smallest genetic engineer• Agrobacterium tumefacians• Dicots• Acetosyringone and Hydroxyacetosyringone• Cancerous proliferation of stem tissue• Ti plasmid (Tumor inducing) >200Kb• T DNA: 15-30 Kb• Genetically engineers the plant cell for its own purposes• A-G system

A-G system• Vir A: Transmembrane protein• Vir A phosphorylates Vir G binds to promoter and activates vir

genes• Vir D1 and D2: Border endonucleases (D2 remain attached)• Vir E: SSB protein• Vir B: transport process• T DNA transported and integrated into host Complementary strand

synthesis

Vector construction• Large size of Ti plasmid – difficult to manipulate• Unique restriction site

2 strategies for construction:The Binary Vector StrategyThe Co-integration Strategy

The Binary Vector Strategy

pBIN19

The Binary Vector Strategy

The Co-integration Strategy

Production of transformed cells