Standardizing cassette-based deep mutagenesis by Golden Gate assembly

Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system-specific methodology. Here we present a standardized Golden Gate method for building user-defined libraries. We demonstrate that a 25 mu L reaction, using 40 fmol of input DNA, can generate a library on the order of 1 x 106 members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real-world effectiveness by building custom, user-defined libraries on the order of 104-107 unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general-use destination vectors. A standardized protocol for cassette-based mutagenesis by Golden Gate is presented. Number of transformants scale with amount of input DNA at high incorporation efficiency.image

Automated summary

A standardized Golden Gate method for building user-defined libraries is presented, allowing the construction of large, deeply mutagenized protein encoding libraries. This method is efficient and capable of generating libraries with a high number of protein encoding variants. It can be applied to different protein engineering systems and provides general-use destination vectors and detailed protocols for other researchers.