A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering.

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 µl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.

Avoiding and controlling double transformation artifacts.

This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from 'double transformants' -- i.e. cells transformed with, or infected by, two different plasmids simultaneously. These seem to have been the source of past artifacts and, as demonstrated here, are far more common than generally recognized. By following standard protocols for cloning, plasmid isolation, subcloning, in combination with functional assays, the presence of such artifacts can be ruled out. This protocol needs to be applied for any new variant isolated from heterogeneous gene repertoires, and in particular for variants isolated by selection for either enzymatic activity, or binding.

The promise and peril of continuous in vitro evolution.

Experimental evolution methods can be used to address and illuminate issues central to the understanding of evolutionary theory. One of the most powerful of these methods involves the in vitro evolution of nucleic acid enzymes, taking advantage of the direct relationship between the genotype of a nucleic acid sequence and the phenotype of its associated catalytic function. This review and commentary focuses on the past, present, and future potential of systems for the continuous in vitro evolution of nucleic acid enzymes as tools for modeling evolutionary processes in biology. It offers a candid appraisal of both the strengths and the limitations of these systems.