A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.

Generating combinatorial libraries of specific sets of mutations are essential for addressing protein engineering questions involving contingency in molecular evolution, epistatic relationships between mutations, as well as functional antibody and enzyme engineering. Here we present optimization of a combinatorial mutagenesis method involving template-based nicking mutagenesis, which allows for the generation of libraries with >99% coverage for tens of thousands of user-defined variants. The non-optimized method resulted in low library coverage, which could be rationalized by a model of oligonucleotide annealing bias resulting from the nucleotide mismatch free-energy difference between mutagenic oligo and template. The optimized method mitigated this thermodynamic bias using longer primer sets and faster annealing conditions. Our updated method, applied to two antibody fragments, delivered between 99.0% (32451/32768 library members) to >99.9% coverage (32757/32768) for our desired libraries in 2 days and at an approximate 140-fold sequencing depth of coverage.

Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by Taq DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated covalently closed circular plasmids. It was later shown that because very little primer is used in the procedure, resuspended oligo pools, which normally require amplification before use, can be used directly in the mutagenesis procedure. Because oligo pools can contain tens of thousands of unique oligos, this enables the construction of libraries of tens of thousands of user-defined mutations in a single-pot mutagenesis reaction, which significantly improves the utility of NM as described below. Use of oligo pools afford an economically advantageous approach to mutagenic experiments. First, oligo pool synthesis is much less expensive per nucleotide synthesized than conventional synthesis. Second, a mixed pool may be generated and used for mutagenesis of multiple different genes. To use the same oligo-pool for mutagenesis of a variety of genes, the user must only quantify the fraction of the oligo-pool specific to her mutagenic experiment and adjust the volume and effective concentration of the oligo-pool for use in nicking mutagenesis.

A high-throughput mutation screen dissects the mechanistic basis of enzyme activity

Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system specific methodology. Here we benchmark a broadly applicable Golden Gate method for building user-defined libraries. We demonstrate that a 25 μl reaction, using 40 fmol of input DNA, can generate a library on the order of 1x106members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real-world effectiveness by building custom, user-defined libraries on the order of 104to 107unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general-use destination vectors.