Golden Gate Assembly: Assembly of Cassette Plasmids

  • The simplest way to assemble a cassette is to include one part of each Type in a BsaI assembly. The plasmid pYTK095 included in yeast toolkit serve as the canonical “vectors”, and accordingly have an sfGFP expression dropout for Green/white screening.

Cassette plasmids contain a complete transcriptional unit (TU), and can be transformed directly into target host. Plasmids at this stage typically confer ampicillin resistance(pYTK095) in E. coli. For example, the preassembled vector pYTK095 is assembled from ConLS′ (Type 1), sfGFP dropout (Type 234), ConRE′(Type 5), AmpR-ColE1 (Type 678). A transcriptional unit (promoter, coding sequence, terminator) can be assembled into this vector, replacing the BsaI-flanked sfGFP dropout. When you performing this, you need ConL (Type 1,ConLS/L1/L2/L3/L4/L5), Promoter (Type 2), Coding sequence (Type 3),Terminator (Type 4), ConRE′(Type 5, ConR1/R2/R3/R4/R5/RE), AmpR-ColE1 (Type 678).

Assembly_of_cassette_plasmids.jpg

-- Main.PengGeng - 25 Aug 2016

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Topic revision: r1 - 25 Aug 2016 - 20:03:08 - Main.PengGeng