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The success of ultrahigh-throughput screening experiments in directed evolution or functional metagenomics strongly depends on the availability of efficient technologies for the quantitative testing of a large number of variants. With advanced robotics, libraries of up to 10

USER friendly DNA recombination (USERec) is based on near homology-independent recombination of DNA fragments (~40-400 bp) that are efficiently reassembled into full-length genes, proven for up to ten fragments. USERec requires a minimal crossover sequence (a 5'-AN(4-8)T-3' motif) of the fragments that can be implemented wherever structural or functional comparisons suggest a fragment boundary. The greatly reduced sequence constraints of this method facilitate directional assembly of gene fragments for applications such as exon or domain shuffling, loop grafting, reassembly of natural modular biosynthetic assembly lines, and rearrangement of structurally (but not sequence) homologous proteins.

Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets.

Cell-free expression systems provide straightforward access from genes to the corresponding proteins, involving fewer handling steps than in vivo procedures. A quick procedure to assemble a gene of interest into a linear DNA template together with 3'- and 5'-untranslated regions using a coupled uracil-excision-ligation strategy based on USER Enzyme and T4 DNA ligase. This methodology will be useful for repeated cycles of expression and in vitro selection, in which gene libraries are repeatedly assembled and their products and templates regenerated.

SNAP display is based on the covalent reaction of the DNA repair protein AGT (O(6)-alkylguanine DNA alkyltransferase, the "SNAP-tag") with its substrate benzylguanine (BG). Linear, BG-labelled template DNA is encapsulated in water-in-oil emulsion droplets with a diameter of a few micrometres (i.e. 1 mL of emulsion contains ∼10(10) compartments). Each droplet contains only a single DNA copy, which is transcribed and translated in vitro. The expressed AGT fusion proteins attach to their coding DNA via the BG label inside the droplet, which ensures that a specific genotype-phenotype linkage is established. Subsequently, the emulsion is broken and protein-DNA conjugates, which constitute a DNA-tagged protein library, selected via affinity panning. This method will prove a useful addition to the array of in vitro display systems, distinguished by the stability of DNA as the coding nucleic acid and the covalent link between gene and protein.