Systematic molecular evolution enables robust biomolecule discovery.

Evolution occurs when selective pressures from the environment shape inherited variation over time. Within the laboratory, evolution is commonly used to engineer proteins and RNA, but experimental constraints have limited the ability to reproducibly and reliably explore factors such as population diversity, the timing of environmental changes and chance on outcomes. We developed a robotic system termed phage- and robotics-assisted near-continuous evolution (PRANCE) to comprehensively explore biomolecular evolution by performing phage-assisted continuous evolution in high-throughput. PRANCE implements an automated feedback control system that adjusts the stringency of selection in response to real-time measurements of each molecular activity. In evolving three distinct types of biomolecule, we find that evolution is reproducibly altered by both random chance and the historical pattern of environmental changes. This work improves the reliability of protein engineering and enables the systematic analysis of the historical, environmental and random factors governing biomolecular evolution.

Single-cell activity screening in microfluidic droplets.

Water-in-oil emulsion droplets can be used as microcompartments to contain single cells that can be subjected to activity assays in this format. Microfluidic devices produce droplets at > kHz rates and can be coupled to modules to, e.g., add reagents, incubate or measure analyte concentration optically (with sensitivities as low as 2nM). The range of optical assays includes fluorescence and absorbance detection and examples for the use of these technologies for ultrahigh-throughput sorting in directed evolution and functional metagenomics are described.

High-throughput droplet-based microfluidics for directed evolution of enzymes.

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user-defined goal) coupled with droplet-based microfluidics allows the detailed analysis of millions of enzyme variants on ultra-short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet-based microfluidics, made in this context towards the high-throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell-based assays, as in the case of directed evolution experimentations.

Mini Photobioreactors for in Vivo Real-Time Characterization and Evolutionary Tuning of Bacterial Optogenetic Circuit.

The current standard protocols for characterizing the optogenetic circuit of bacterial cells using flow cytometry in light tubes and light exposure of culture plates are tedious, labor-intensive, and cumbersome. In this work, we engineer a bioreactor with working volume of ∼10 mL for in vivo real-time optogenetic characterization of E. coli with a CcaS-CcaR light-sensing system. In the bioreactor, optical density measurements, reporter protein fluorescence detection, and light input stimuli are provided by four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth and gene expression without human intervention. We measure gene expression during cell growth with different organic substrates (glucose, succinate, acetate, pyruvate) as carbon sources in minimal medium and demonstrate evolutionary tuning of the optogenetic circuit by serial dilution passages.

In vivo continuous directed evolution.

The development and application of methods for the laboratory evolution of biomolecules has rapidly progressed over the last few decades. Advancements in continuous microbe culturing and selection design have facilitated the development of new technologies that enable the continuous directed evolution of proteins and nucleic acids. These technologies have the potential to support the extremely rapid evolution of biomolecules with tailor-made functional properties. Continuous evolution methods must support all of the key steps of laboratory evolution - translation of genes into gene products, selection or screening, replication of genes encoding the most fit gene products, and mutation of surviving genes - in a self-sustaining manner that requires little or no researcher intervention. Continuous laboratory evolution has been historically used to study problems including antibiotic resistance, organismal adaptation, phylogenetic reconstruction, and host-pathogen interactions, with more recent applications focusing on the rapid generation of proteins and nucleic acids with useful, tailor-made properties. The advent of increasingly general methods for continuous directed evolution should enable researchers to address increasingly complex questions and to access biomolecules with more novel or even unprecedented properties.

SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution.

The de novo synthesis of genes is becoming increasingly common in synthetic biology studies. However, the inherent error rate (introduced by errors incurred during oligonucleotide synthesis) limits its use in synthesising protein libraries to only short genes. Here we introduce SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enabling the efficient synthesis of large genes for use directly in functional screening. First, we demonstrate an accurate gene synthesis method by synthesising and directly screening (without pre-selection) a 747 bp gene for green fluorescent protein (yielding 85% fluorescent colonies) and a larger 1518 bp gene (a monoamine oxidase, producing 76% colonies with full catalytic activity, a 4-fold improvement over previous methods). Secondly, we show that SpeedyGenes can accommodate multiple and combinatorial variant sequences while maintaining efficient enzymatic error correction, which is particularly crucial for larger genes. In its first application for directed evolution, we demonstrate the use of SpeedyGenes in the synthesis and screening of large libraries of MAO-N variants. Using this method, libraries are synthesised, transformed and screened within 3 days. Importantly, as each mutation we introduce is controlled by the oligonucleotide sequence, SpeedyGenes enables the synthesis of large, diverse, yet controlled variant sequences for the purposes of directed evolution.

CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics.

We present a high-throughput droplet-based microfluidic analysis/screening platform for directed evolution of CotA laccase: droplet-based microfluidic modules were combined to develop an efficient system that allows cell detection and sorting based on the enzymatic activity. This platform was run on two different operating modes: the "analysis" mode allowing the analysis of the enzymatic activity in droplets at very high rates (>1000 Hz) and the "screening" mode allowing sorting of active droplets at 400 Hz. The screening mode was validated for the directed evolution of the cytoplasmic CotA laccase from B. subtilis, a potential interesting thermophilic cathodic catalyst for biofuel cells. Single E. coli cells expressing either the active CotA laccase (E. coli CotA) or an inactive frameshifted variant (E. coli ΔCotA) were compartmentalized in aqueous droplets containing expression medium. After cell growth and protein expression within the droplets, a fluorogenic substrate was "picoinjected" in each droplet. Fluorescence-activated droplet sorting was then used to sort the droplets containing the desired activity and the corresponding cells were then recultivated and identified using colorimetric assays. We demonstrated that E. coli CotA cells were enriched 191-fold from a 1 : 9 initial ratio of E. coli CotA to E. coli ΔCotA cells (or 437-fold from a 1 : 99 initial ratio) using a sorting rate of 400 droplets per s. This system allows screening of 10(6) cells in only 4 h, compared to 11 days for screening using microtitre plate-based systems. Besides this low error rate sorting mode, the system can also be used at higher throughputs in "enrichment" screening mode to make an initial purification of a library before further steps of selection. Analysis mode, without sorting, was used to rapidly quantify the activity of a CotA library constructed using error-prone PCR. This mode allows analysis of 10(6) cells in only 1.5 h.

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule.

Visual automated macromolecular model building.

Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user.

A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets.

The enormous reduction of assay volume afforded by compartmentalization into picolitre water-in-oil droplets is an exciting prospect for high-throughput biology. Maintaining the activity of encapsulated proteins is critical for experimental success, for example in in vitro directed evolution, where protein variants are expressed in droplets to identify mutants with improved properties. Here, we present a simple and rapid method to quantitatively compare concentrations of fluorescent molecules in microdroplets. This approach allows an assessment of different emulsification procedures and several oil/surfactant mixtures for biochemical compatibility, in particular in vitro protein expression. Based on determining droplet fluorescence vs. droplet diameter, the method uses the gradient of such curves as a 'concentration correlation coefficient' (CCC) that is directly proportional to fluorophore concentration. Our findings suggest that generation of droplets using a microfluidic flow-focusing device gave no more protein expression than droplet production by the bulk methods of vortexing and homogenizing. The choice of oil/surfactant, however, was found to be critical for protein expression and even encapsulation of purified protein, highlighting the importance of careful selection of these components when carrying out biochemical experiments in droplets. This methodology will serve as a quantitative test for the rapid optimization of droplet-based experiments such as in vitro protein expression or enzymatic assays.

A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

Microfluidic landscapes for evolution.

Evolution at its heart is an iterative algorithm composed of three steps: selection, amplification and mutagenesis. This algorithm can be applied to complex inputs such as populations of whole organisms and viruses, or mixtures of bare nucleic acids and proteins. The output is the same: evolutionary adaptation of new and improved function subject to selection. Recent breakthroughs in microfluidic technology have introduced automation and process monitoring to in vitro evolution, and reproducible preparation of emulsions and other multi-phase reaction landscapes. It is at this intersection of compartmentalization and in vitro evolution where miniaturization is again redefining experimental design in contemporary chemistry and biology.

Microfluidic compartmentalized directed evolution.

Directed evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 +/- 0.8 microm radius) with high throughput (10(7)-10(8) droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channel. This device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin. Each molecule in the population had the opportunity to undergo 10(8)-fold selective amplification within its respective compartment. Then the progeny RNAs were harvested and used to seed new compartments. During five rounds of this procedure, the enzymes acquired mutations that conferred resistance to neomycin and caused some enzymes to become dependent on neomycin for optimal activity.

Programming cells by multiplex genome engineering and accelerated evolution.

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

Lab-on-a-chip in vitro compartmentalization technologies for protein studies.

In vitro compartmentalization (IVC) is a powerful tool for studying protein-protein reactions, due to its high capacity and the versatility of droplet technologies. IVC bridges the gap between chemistry and biology as it enables the incorporation of unnatural amino acids with modifications into biological systems, through protein transcription and translation reactions, in a cell-like microdrop environment. The quest for the ultimate chip for protein studies using IVC is the drive for the development of various microfluidic droplet technologies to enable these unusual biochemical reactions to occur. These techniques have been shown to generate precise microdrops with a controlled size. Various chemical and physical phenomena have been utilized for on-chip manipulation to allow the droplets to be generated, fused, and split. Coupled with detection techniques, droplets can be sorted and selected. These capabilities allow directed protein evolution to be carried out on a microchip. With further technological development of the detection module, factors such as addressable storage, transport and interfacing technologies, could be integrated and thus provide platforms for protein studies with high efficiency and accuracy that conventional laboratories cannot achieve.

Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist.

We have developed an automated SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process that allows the execution of in vitro selection cycles without any direct manual intervention steps. The automated selection protocol is designed to provide for high flexibility and versatility in terms of choice of buffers and reagents as well as stringency of selection conditions. Employing the automated SELEX process, we have identified RNA aptamers to the mirror-image configuration (d-peptide) of substance P. The peptide substance P belongs to the tachykinin family and exerts various biologically important functions, such as peripheral vasodilation, smooth muscle contraction and pain transmission. The aptamer that was identified most frequently was truncated to the 44mer SUP-A-004. The mirror-image configuration of SUP-A-004, the so-called Spiegelmer, has been shown to bind to naturally occurring l-substance P displaying a K(d) of 40 nM and to inhibit (IC50 of 45 nM) l-substance P-mediated Ca2+ release in a cell culture assay.

A scalable high-throughput chemical synthesizer.

A machine that employs a novel reagent delivery technique for biomolecular synthesis has been developed. This machine separates the addressing of individual synthesis sites from the actual process of reagent delivery by using masks placed over the sites. Because of this separation, this machine is both cost-effective and scalable, and thus the time required to synthesize 384 or 1536 unique biomolecules is very nearly the same. Importantly, the mask design allows scaling of the number of synthesis sites without the addition of new valving. Physical and biological comparisons between DNA made on a commercially available synthesizer and this unit show that it produces DNA of similar quality.

A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases.

BACKGROUND: Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples. RESULTS: Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450cam by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities. CONCLUSIONS: The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.