Polishing the craft of genetic diversity creation in directed evolution

Abstract

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

“…13| Use a library generation method of choice 3 ‘ 4 to prepare a randomized G0I pool and subclone the pool into an appropriate destination vector (at least 1 μρ vector).…”

“…The details of library design and generation have been covered in many other places 3 · 4 and are beyond the scope of this protocol. We will, therefore, only briefly describe the key points for molecular cloning of T7 RNAP and tRNA pools in appropriate plasmid vectors.…”

“…In a typical directed evolution experiment, genetic diversity is first created via either random or targeted mutagenesis, or through random recombination between a number of evolutionary related genetic elements 3,4 . The size of the resulting library can vary based on the method, but is typically on the order of 10 6 or greater, which is much larger than the number of mutations that typically exist in a similar natural population.…”

Compartmentalized Self‐Replication for Evolution of a DNA Polymerase

“…Consider improving the library design or switching to a different a library generation method altogether (Gillam et al, 2014;Tee & Wong, 2013). First, since the fraction of active variant in the pool affects ePCR and Recovery PCR yields, the Recovery PCR yield can be used as a proxy for the enrichment progress.…”

“…Thus, library generation is generally beyond the scope of this unit, and we refer the reader to other resources (Gillam, Copp, & Ackerley, 2014;Tee & Wong, 2013). While library generation and diversity are of exquisite importance for a selection experiment, the particular libraries that will be plumbed are as diverse as the potential functions that will be selected for.…”