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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

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Published on: July 25, 2013

Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering.

John N Milligan¹, Daniel J Garry²

¹The Department of Molecular Biosciences, The University of Texas at Austin, 2506 Speedway STOP A5000, Austin, TX, 78712, USA. john.milligan@utexas.edu.

Methods in Molecular Biology (Clifton, N.J.)

|September 28, 2016

Summary

This summary is machine-generated.

This study introduces Shuffle Optimizer, a free Python program that enhances DNA homology for protein engineering. The protocol details DNA shuffling, primer design, and library construction for creating protein variants.

Keywords:

Codon optimization Computer program DNA shuffling Protein engineering Protein library Python Random chimeragenesis on transient templates (RACHITT)Staggered extension process (StEP)

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Area of Science:

Biochemistry
Molecular Biology
Bioinformatics

Background:

DNA shuffling is a key technique for generating protein variant libraries in protein engineering.
Existing methods can be complex and require specific computational tools.

Purpose of the Study:

To present a user-friendly protocol and a free computer program, Shuffle Optimizer, for DNA shuffling.
To improve the nucleotide homology between DNA fragments for efficient shuffling without altering amino acid sequences.

Main Methods:

Development of the Shuffle Optimizer program using Python.
Detailed protocols for optimal primer design and DNA library construction.
Implementation of a small-volume ultrasonicator method for DNA shearing.
Method for reassembling sheared DNA fragments, library recovery, and cloning.

Main Results:

Shuffle Optimizer successfully increases nucleotide homology between DNA fragments.
The protocol provides a comprehensive workflow from DNA shearing to library cloning.
The program and methods are designed for ease of use and accessibility.

Conclusions:

The Shuffle Optimizer program and accompanying protocols offer a valuable resource for researchers performing DNA shuffling.
These tools facilitate the creation of diverse protein variant libraries for protein engineering applications.
The presented methods are applicable to various nucleotide homology-dependent shuffling techniques.