Viktor Stein

Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets

We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.

Synthetic protein switches: design principles and applications

Protein switches are ubiquitous in biological signal transduction systems, enabling cells to sense and respond to a variety of molecular queues in a rapid, specific, and integrated fashion. Analogously, tailor-engineered protein switches with custom input and output functions have become invaluable research tools for reporting on distinct physiological states and actuating molecular functions in real time and in situ. Here, we analyze recent progress in constructing protein-based switches while assessing their potential in the assembly of defined signaling motifs. We anticipate such systems will ultimately pave the way towards a new generation of molecular diagnostics and facilitate the construction of artificial signaling systems that operate in parallel to the signaling machinery of a host cell for applications in synthetic biology.

A Covalent Chemical Genotype–Phenotype Linkage for in vitro Protein Evolution

Reference LIP-ARTICLE-2008-008doi:10.1002/cbic.200700459View record in Web of Science Record created on 2008-06-09, modified on 2017-05-12

An efficient method to assemble linear DNA templates for in vitro screening and selection systems

A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in ∼90 min. Assembly is achieved using a coupled uracil excision–ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 1011 molecules can be efficiently assembled and purified in reaction volumes of 100 μl. The method is exemplified with the gene coding for a mutant version of O6-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5′- and 3′-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles.

Protease-based synthetic sensing and signal amplification

Significance Synthetic biology envisages the creation of custom-based signaling by means of modular plug-and-play. This concept has primarily been realized in the construction of synthetic gene circuits. However, all real-time events in biology are processed by protein-based sensing and signal transducing systems; yet, the systematic bottom-up design of protein-based signaling systems remains elusive to date. Here we report a strategy for construction of modular protein switches based on artificially autoinhibited proteases whose activity can be modulated by specific proteolysis, ligand binding, or protein–protein interactions. We demonstrate that such protease-based ligand receptors or signal transducers can be assembled into different types of integrated signal sensing and amplification circuits that, in principle, can be connected to any biological process. The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor’s mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.

Research Article: SPORCalc: A development of a database analysis that provides putative metabolic enzyme reactions for ligand-based drug design

Understanding both the enzyme reactions that contribute to intermediate metabolism and the biochemical fate of candidate therapeutic and toxic agents are essential for drug design. Traditional metabolic databases indicate whether reactions have been observed but do not provide the likelihoods of reactions occurring, for example those of mixed function oxygenases and oxidases, during phase I metabolism. The desire for more quantitative predictions motivated the development of the recently introduced Substrate Product Occurrence Ratio Calculator (SPORCalc) that identifies metabolically labile atom positions in candidate compounds. This paper describes a further development and provides a clearer explanation of SPORCalc for the computational pharmacology, medicinal chemistry and drug design communities interested in metabolic prediction of xenobiotics using chemical databases of biotransformations. Examples of reaction centre detection in Metabolite are described followed by a demonstration of almokalant, an anti-arrhythmic agent, undergoing phase I metabolism. In general, occurrence ratio (OR) values are calculated throughout a compound and its transformed metabolites to give propensity (p) values at each atom position. The OR values from substrates and products in the database are essential for addition and elimination reactions. For almokalant, the resulting p values ranged from 10(-1) to 10(-5) and their order of magnitude reflected the known and experimentally observed metabolites. SPORCalc depends entirely on the level of detail from isoform- or species-specific reaction classes in Metabolite. Labile atom positions (sites of metabolism) are identified in both the candidate compound and its metabolites. In general, the likelihood of one enzyme isoform-dependent reaction occurring relative to another and the putative metabolic routes from different isoforms can be investigated. SPORCalc can be developed further to include suitable three-dimensional, structure-activity and physiochemical information.

SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof‐of‐concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer‐like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water‐in‐oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP‐tag fusion. The covalent bond between DNA and the SNAP‐tag is created by reaction with dendrimer‐bound benzylguanine (BG). The ability to form dendrimer‐like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400‐fold enrichments. The comparison of mono‐ and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25‐fold. Our data establish a multivalent format for SNAP‐display based on dendrimer‐like DNA as the first in vitro display system with defined tailor‐made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered.

Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification

In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1 h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3–10 ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification.

Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform

Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules.

Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.