Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering.
DNA shuffling is a powerful tool to develop libraries of variants for protein engineering. Here, we present a protocol to use our freely available and easy-to-use computer program, Shuffle Optimizer. Shuffle Optimizer is written in the Python computer language and increases the nucleotide homology between two pieces of DNA desired to be shuffled together without changing the amino acid sequence. In addition we also include sections on optimal primer design for DNA shuffling and library construction, a small-volume ultrasonicator method to create sheared DNA, and finally a method to reassemble the sheared fragments and recover and clone the library. The Shuffle Optimizer program and these protocols will be useful to anyone desiring to perform any of the nucleotide homology-dependent shuffling methods.
The Department of Molecular Biosciences, The University of Texas at Austin, 2506 Speedway STOP A5000, Austin, TX, 78712, USA. john.milligan@utexas.edu.
John N
JN
Milligan
The Department of Molecular Biosciences, The University of Texas at Austin, 2506 Speedway STOP A5000, Austin, TX, 78712, USA. djgarry@utexas.edu.
Daniel J
DJ
Garry
D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D012984:Software
D017931:DNA Primers
Codon optimization; Computer program; DNA shuffling; Protein engineering; Protein library; Python; Random chimeragenesis on transient templates (RACHITT); Staggered extension process (StEP)