Joseph P. Torella

Conformational transitions in DNA polymerase I revealed by single-molecule FRET

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.

Efficient solar-to-fuels production from a hybrid microbial–water-splitting catalyst system

Significance Renewable-fuels generation has emphasized water splitting to produce hydrogen and oxygen. For accelerated technology adoption, bridging hydrogen to liquid fuels is critical to the translation of solar-driven water splitting to current energy infrastructures. One approach to establishing this connection is to use the hydrogen from water splitting to reduce carbon dioxide to generate liquid fuels via a biocatalyst. We describe the integration of water-splitting catalysts comprised of earth-abundant components to wild-type and engineered Ralstonia eutropha to generate biomass and isopropyl alcohol, respectively. We establish the parameters for bacterial growth conditions at low overpotentials and consequently achieve overall efficiencies that are comparable to or exceed natural systems. Photovoltaic cells have considerable potential to satisfy future renewable-energy needs, but efficient and scalable methods of storing the intermittent electricity they produce are required for the large-scale implementation of solar energy. Current solar-to-fuels storage cycles based on water splitting produce hydrogen and oxygen, which are attractive fuels in principle but confront practical limitations from the current energy infrastructure that is based on liquid fuels. In this work, we report the development of a scalable, integrated bioelectrochemical system in which the bacterium Ralstonia eutropha is used to efficiently convert CO2, along with H2 and O2 produced from water splitting, into biomass and fusel alcohols. Water-splitting catalysis was performed using catalysts that are made of earth-abundant metals and enable low overpotential water splitting. In this integrated setup, equivalent solar-to-biomass yields of up to 3.2% of the thermodynamic maximum exceed that of most terrestrial plants. Moreover, engineering of R. eutropha enabled production of the fusel alcohol isopropanol at up to 216 mg/L, the highest bioelectrochemical fuel yield yet reported by >300%. This work demonstrates that catalysts of biotic and abiotic origin can be interfaced to achieve challenging chemical energy-to-fuels transformations.

Tailored fatty acid synthesis via dynamic control of fatty acid elongation

Medium-chain fatty acids (MCFAs, 4–12 carbons) are valuable as precursors to industrial chemicals and biofuels, but are not canonical products of microbial fatty acid synthesis. We engineered microbial production of the full range of even- and odd-chain–length MCFAs and found that MCFA production is limited by rapid, irreversible elongation of their acyl-ACP precursors. To address this limitation, we programmed an essential ketoacyl synthase to degrade in response to a chemical inducer, thereby slowing acyl-ACP elongation and redirecting flux from phospholipid synthesis to MCFA production. Our results show that induced protein degradation can be used to dynamically alter metabolic flux, and thereby increase the yield of a desired compound. The strategy reported herein should be widely useful in a range of metabolic engineering applications in which essential enzymes divert flux away from a desired product, as well as in the production of polyketides, bioplastics, and other recursively synthesized hydrocarbons for which chain-length control is desired.

Identifying Molecular Dynamics in Single-Molecule FRET Experiments with Burst Variance Analysis

Histograms of single-molecule Förster resonance energy transfer (FRET) efficiency are often used to study the structures of biomolecules and relate these structures to function. Methods like probability distribution analysis analyze FRET histograms to detect heterogeneities in molecular structure, but they cannot determine whether this heterogeneity arises from dynamic processes or from the coexistence of several static structures. To this end, we introduce burst variance analysis (BVA), a method that detects dynamics by comparing the standard deviation of FRET from individual molecules over time to that expected from theory. Both simulations and experiments on DNA hairpins show that BVA can distinguish between static and dynamic sources of heterogeneity in single-molecule FRET histograms and can test models of dynamics against the observed standard deviation information. Using BVA, we analyzed the fingers-closing transition in the Klenow fragment of Escherichia coli DNA polymerase I and identified substantial dynamics in polymerase complexes formed prior to nucleotide incorporation; these dynamics may be important for the fidelity of DNA synthesis. We expect BVA to be broadly applicable to single-molecule FRET studies of molecular structure and to complement approaches such as probability distribution analysis and fluorescence correlation spectroscopy in studying molecular dynamics.

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two-state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis-testing models of conformational dynamics against experimental data.

Two- and three-input TALE-based AND logic computation in embryonic stem cells

Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals.

Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.

Single‐Molecule DNA Biosensors for Protein and Ligand Detection

Transcription factors (TFs) are sequence-specific DNA-binding proteins that control much of gene expression. TFs are natural biosensors and switches, translating chemical and physical signals (temperature shifts, light exposure, chemical concentrations, redox status) into transcriptional changes by modulating the binding of RNA polymerase to promoter DNA. Since changes in TF levels underlie fundamental biological processes such as DNA repair and cell-cycle progression, alterations in the levels of active TFs both lead to and indicate disease; for example, mutations in transcription factor p53 contribute to the rapid growth of cancer cells and, owing to their prevalence (p53 is mutated in roughly 50% of all human tumors), they have served as cancer biomarkers. Thus, methods for the sensitive detection and quantitation of TFs provide both fundamental information about gene regulation and a platform for diagnostics. TF detection often involves gel-based assays and Western blotting; although helpful in characterizing TF–DNA interactions, these assays are tedious, expensive, and qualitative, and consume large quantities of sample. Enzyme-linked immunosorbent assays (ELISAs) are more sensitive and offer higher throughput, but they require many preparation and signal-amplification steps for the detection of lowabundance TFs. Amplification is also required in the proximity-based ligation assay, making it incompatible with TF detection in living cells and diagnostic settings that demand results within minutes. An additional TF detection assay is based on fluorescence resonance energy transfer (FRET) between two doublestranded DNA (dsDNA) fragments containing fluorescently labeled single-stranded complementary overhangs (“molecular beacons”). In the presence of TF, the DNAs associate, resulting in donor fluorophore quenching as a result of FRET. This assay still requires significant amounts of sample and cannot detect low-abundance TFs; and because of the short dynamic range of FRET (1–10 nm), it also requires close proximity among the fluorophore, the quencher, and the protein–DNA interface, increasing the likelihood of steric interference with protein–DNA binding and complicating sensor design. Moreover, placing the fluorophore and the quencher on either side of the protein-binding site (usually 15–30 base pairs (bp) in length) on DNA results in very low FRET signals for most TFs. Here, we use alternating-laser excitation (ALEX) spectroscopy to detect TFs and small molecules by means of the TF-dependent coincidence of fluorescently labeled DNA. Like the molecular-beacon assay, our method is based on TFdriven DNA association, is rapid, and requires no amplification. However, our assay can detect pm levels of TFs in small amounts of sample, and it is FRET-independent, bypassing the need to optimize fluorophore position or know the structural details of TF–DNA binding; this flexibility in labeling ensures unperturbed TF–DNA binding. Using ALEX, we demonstrate TF and small-molecule detection, assay multiplexing, and suitability for analysis of complex biological samples. In our assay (Figure 1a,b), the full DNA-binding site for a TF is split in two (as in Ref. [5]): the left half-site (H1) and the right half-site (H2). Each site contains half of the TF-binding determinants and short, complementary 3’-overhangs. H1 is labeled with a “green” fluorophore (“G”) to give half-site H1, whereas H2 is labeled with a spectrally distinct “red” fluorophore (“R”) to give H2. In the absence of TF and at DNA concentrations of roughly 10–100 pm, H1 and H2 diffuse independently and associate only transiently. In contrast, in the presence of a TF that binds to the fully assembled DNA site, H1 and H2 diffuse as a complex (H1TF-H2; Figure 1a, bottom). We detect TF-dependent DNA coincidence using ALEX spectroscopy, wherein single molecules are excited by two lasers in an alternating fashion, with each laser capable of directly exciting either a G or a R fluorophore. ALEX allows molecular sorting on two-dimensional histograms of apparent FRET efficiency E* (a fluorescence ratio that reports on interfluorophore proximity) and probe stoichiometry S (a fluorescence ratio that reports on molecular stoichiometry). A search for all R-labeled molecules (i.e., G–R molecules [*] Dr. K. Lymperopoulos, R. Crawford, J. P. Torella, Dr. M. Heilemann, Dr. L. C. Hwang, S. J. Holden, Dr. A. N. Kapanidis Biological Physics Research Group, Department of Physics University of Oxford, Clarendon Laboratory Parks Road, Oxford, OX1 3PU (United Kingdom) E-mail: a.kapanidis1@physics.ox.ac.uk Dr. K. Lymperopoulos Current address: BioQuant Institute, Cellnetworks Cluster Ruprecht-Karls Universit t Heidelberg 69120 Heidelberg (Germany)

Unique nucleotide sequence–guided assembly of repetitive DNA parts for synthetic biology applications

Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies—for example, repeated terminator and insulator sequences—that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)–guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2–3 d. If the DNA parts must be generated from scratch, an additional 2–5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques.