Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.
|State||Published - 20 Jul 2015|
Bibliographical noteFunding Information:
This work was supported by the Max-Planck-Society, the LOEWE Research Cluster SynChemBio from the state of Hessen, Germany; and the Israeli Science Foundation (grant 286/13). C.G.A.R is holder of a SYNMIKRO postdoctoral fellowship awarded by the LOEWE program of the state of Hessen, Germany.
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