Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene—or gene circuit function—that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks

High-Throughput Screening of Enzymes by Retroviral Display Using Droplet-Based Microfluidics

SummaryDuring the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require posttranslational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a non-active control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated