THORSTEN PFIRRMANN*, REBECCA WAGNER, THOMAS HOLLEMANN
*Corresponding author
Martin-Luther University Halle-Wittenberg, Institute for Physiological Chemistry, D-06114 Halle, Germany

Abstract

The ability to rapidly and precisely assemble diverse genetic elements is critical for many fields of modern biology, ranging from research in synthetic biology to molecular ageing. Despite the rapidly dropping prices of synthetic genes there is still a high demand for methods that allow production of recombinant hybrid proteins and methods to mutagenize genes targeted, semi-randomly or randomly, with the aim to produce engineered enzymes with e.g. higher stabilities, altered specificity or novel functions. To satisfy this demand we recently published a method dubbed SOMA (Single-Oligonucleotide Mutagenesis and Cloning Approach). The many advantages of SOMA towards classical cloning and mutagenesis approaches will be discussed here with the aim to promote the method and to make it applicable to a broader community. As a novel application we use the insertion feature of SOMA to generate CRISPR constructs for directional genome editing.

INTRODUCTION 

Gene synthesis has revolutionized molecular biology. Virtually any gene can be synthesized from scratch and can be customized for any specific need. This has changed the work of modern molecular biologists in recent years and will probably change it even more in the future. Molecular biologists will deal with larger DNA fragments to not only clone single genes into expression vectors, but to clone sets of genes into synthetic pathways or to fuse genetic building blocks for their specific needs to engineer customized genes, genetic pathways and even complete synthetic genomes. Despite the dropping prices of gene synthesis it is still important to be able to flexibly modify genetic building blocks, e.g. to introduce mutations such as substitutions or insertions. Additionally, the restriction site independent shuffling of such building blocks, e.g. the exchange of different promoters with synthetic genes of interest, will be critical for scientific progress.
This requires methods for restriction site independent shuffling of genes and domains as well as methods for site-directed- or random-mutagenesis for gene fine-tuning (1). In the ...