66 results found
Chan DTC, Baldwin GS, Bernstein HC, 2023, Revealing the Host-Dependent Nature of an Engineered Genetic Inverter in Concordance with Physiology., Biodes Res, Vol: 5
Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to "model status," synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the "chassis effect." It remains a major challenge to uncover which genome-encoded and biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of genetic circuit performance. This was accomplished using a comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis effect and characterize the performance dynamics of a genetic inverter circuit operating within 6 Gammaproteobacteria. Our results solidify the notion that genetic devices are strongly impacted by the host context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar performance of the genetic inverter, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.
Haines MC, Carling B, Marshall J, et al., 2022, basicsynbio and the BASIC SEVA collection: software and vectors for an established DNA assembly method, Synthetic Biology, Vol: 7, ISSN: 2397-7000
Benns HJ, Storch M, Falco JA, et al., 2022, CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery., Nat Microbiol
Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.
Herisson J, Duigou T, du Lac M, et al., 2022, The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering, NATURE COMMUNICATIONS, Vol: 13
Ledesma Amaro R, Ouldridge T, O'Hare D, et al., 2022, Synthetic biology and bioelectrochemical tools for electrogenetic system engineering, Science Advances, Vol: 8, ISSN: 2375-2548
Synthetic biology research and its industrial applications rely on deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and electrodes), allowing for the direct integration of electronics with biological processes. However, use of electrogenetic systems is limited by poor activity, tunability and standardisation. In this work we developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions using a novel, modular bioelectrochemical device. These genetic and electrochemical tools facilitate the design and improve the performance of electrogenetic systems. Furthermore, the genetic design strategies used can be applied to other redox-responsive promoters to further expand the available tools for electrogenetics.
Dwijayanti A, Storch M, Stan G-B, et al., 2022, A modular RNA interference system for multiplexed gene regulation, Nucleic Acids Research, Vol: 50, ISSN: 0305-1048
The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.
Haines MC, Carling B, Marshall J, et al., 2022, basicsynbio and the BASIC SEVA collection: Software and vectors for an established DNA assembly method
<jats:title>Abstract</jats:title><jats:p>Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore design spaces and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use, while enabling simple hierarchical assemblies through an idempotent format. These features facilitate various applications including pathway engineering, ribosome binding site tuning, fusion protein engineering and multiplexed gRNA expression. In this work we present basicsynbio, an open-source software encompassing a Web App (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://basicsynbio.web.app/">https://basicsynbio.web.app/</jats:ext-link>) and Python Package (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/LondonBiofoundry/basicsynbio">https://github.com/LondonBiofoundry/basicsynbio</jats:ext-link>). With basicsynbio, users can access commonly used BASIC parts and linkers while robustly designing new parts and assemblies with exception handling for common design errors. Users can export sequence data and create build instructions for manual or acoustic liquid-handling platforms. The generation of build instructions relies on the BasicBuild Open Standard which is easily parsed for bespoke workflows and is serializable in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio by assembling a collection of 30 vectors using various sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA collection is compatible with BASIC and Golden Gate using BsaI. It encompasses vectors containing six antibiotic resistance mark
Beal J, Baldwin GS, Farny NG, et al., 2021, Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs, PLoS One, Vol: 16, ISSN: 1932-6203
Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.
Beal J, Farny NG, Haddock-Angelli T, et al., 2020, Robust estimation of bacterial cell count from optical density (vol 3, 512, 2020), COMMUNICATIONS BIOLOGY, Vol: 3
Beal J, Farny NG, Haddock-Angelli T, et al., 2020, Robust estimation of bacterial cell count from optical density, COMMUNICATIONS BIOLOGY, Vol: 3
Storch M, Haines MC, Baldwin GS, 2020, DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology, Synthetic Biology, Vol: 5, Pages: ysaa010-ysaa010, ISSN: 2397-7000
Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site and gene order design space for a three-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50-$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratizing automated DNA assembly.
Beal J, Goñi-Moreno A, Myers C, et al., 2020, The long journey towards standards for engineering biosystems: Are the Molecular Biology and the Biotech communities ready to standardise?, EMBO Reports, Vol: 21, Pages: 1-5, ISSN: 1469-221X
Synthetic biology needs to adopt sound scientific and industry-like standards in order to achieve its ambitious goals of efficient and accurate engineering of biological systems.
Storch M, Haines MC, Baldwin GS, 2020, DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology, Synthetic biology (Oxford, England), Vol: 5, ISSN: 2397-7000
Abstract Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site and gene order design space for a three-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50–$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratizing automated DNA assembly.
Storch M, Dwijayanti A, Mallick H, et al., 2020, BASIC: A Simple and Accurate Modular DNA Assembly Method, DNA CLONING AND ASSEMBLY, Vol: 2205, Pages: 239-253, ISSN: 1064-3745
Bartasun P, Prandi N, Storch M, et al., 2019, The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803, PEERJ, Vol: 7, ISSN: 2167-8359
Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced into Synechocystis sp. PCC 6803. We characteriseda new set of ribosome binding site sequences in Synechocystis sp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested con
Haines M, Storch M, Oyarzun D, et al., 2019, Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR), Synthetic Biology, Vol: 4, Pages: 1-10, ISSN: 2397-7000
In vitro selection of ligand-responsive ribozymes can identify rare, functional sequences from large libraries. While powerful, key caveats of this approach include lengthy and demanding experimental workflows; unpredictable experimental outcomes and unknown functionality of enriched sequences in vivo. To address the first of these limitations we developed Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR). LigASERR is scalable, amenable to automation and requires less time to implement compared to alternative methods. To improve the predictability of experiments, we modelled the underlying selection process, predicting experimental outcomes based on sequence and population parameters. We applied this new methodology and model to the enrichment of a known, in vitro-selected sequence from a bespoke library. Prior to implementing selection, conditions were optimised and target sequence dynamics accurately predicted for the majority of the experiment. In addition to enriching the target sequence, we identified two new, theophylline-activated ribozymes. Notably, all three sequences yielded riboswitches functional in Escherichia coli, suggesting LigASERR and similar in vitro selection methods can be utilised for generating functional riboswitches in this organism.
Girvan P, Teng X, Brooks NJ, et al., 2019, Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications, 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495
Haines MC, Storch M, Oyarzún DA, et al., 2019, Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR), Synthetic biology (Oxford, England), Vol: 4, ISSN: 2397-7000
Abstract In vitro selection of ligand-responsive ribozymes can identify rare, functional sequences from large libraries. While powerful, key caveats of this approach include lengthy and demanding experimental workflows; unpredictable experimental outcomes and unknown functionality of enriched sequences in vivo. To address the first of these limitations, we developed Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR). LigASERR is scalable, amenable to automation and requires less time to implement compared to alternative methods. To improve the predictability of experiments, we modeled the underlying selection process, predicting experimental outcomes based on sequence and population parameters. We applied this new methodology and model to the enrichment of a known, in vitro-selected sequence from a bespoke library. Prior to implementing selection, conditions were optimized and target sequence dynamics accurately predicted for the majority of the experiment. In addition to enriching the target sequence, we identified two new, theophylline-activated ribozymes. Notably, all three sequences yielded riboswitches functional in Escherichia coli, suggesting LigASERR and similar in vitro selection methods can be utilized for generating functional riboswitches in this organism.
Silhan J, Zhao Q, Boura E, et al., 2018, Structural basis for recognition and repair of the 3'-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis, Nucleic Acids Research, Vol: 46, Pages: 11980-11989, ISSN: 0305-1048
NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3'-phosphate and other 3'-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3'-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3'-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3'-phosphatase NExo bound to DNA in the presence and absence of a 3'-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3'-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.
Girvan P, Teng X, Brooks N, et al., 2018, Redox kinetics of the amyloid-β-Cu complex and its biological implications, Biochemistry, Vol: 57, Pages: 6228-6233, ISSN: 1520-4995
The ability of the amyloid-β peptide to bind to redox active metals and act as a source of radical damage in Alzheimer’s disease has been largely accepted as contributing to the disease’s pathogenesis. However, a kinetic understanding of the molecular mechanism, which underpins this radical generation, has yet to be reported. Here we use a sensitive fluorescence approach, which reports on the oxidation state of the metal bound to the amyloid-β peptide and can therefore shed light on the redox kinetics. We confirm that the redox goes via a low populated, reactive intermediate and that the reaction proceeds via the Component I coordination environment rather than Component II. We also show that while the reduction step readily occurs (on the 10 ms time scale) it is the oxidation step that is rate-limiting for redox cycling.
Beal J, Haddock-Angelli T, Baldwin G, et al., 2018, Quantification of bacterial fluorescence using independent calibrants, PLoS ONE, Vol: 13, ISSN: 1932-6203
Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.
Storch M, Casini A, Mackrow B, et al., 2016, BASIC: A Simple and Accurate Modular DNA Assembly Method., Methods Mol Biol, Vol: 1472, Pages: 79-91
Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts . The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round .
Webb AJ, Kelwick R, Doenhoff MJ, et al., 2016, A protease-based biosensor for the detection of schistosome cercariae, Scientific Reports, Vol: 6, ISSN: 2045-2322
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses anddeath. Rapid and cost-effective approaches to detect parasites are needed, especially inresource-limited settings. A common signature of parasitic diseases is the release of specificproteases by the parasites at multiple stages during their life cycles. To this end, weengineered several modular Escherichia coli and Bacillus subtilis whole-cell-basedbiosensors which incorporate an interchangeable protease recognition motif into theirdesigns. Herein, we describe how several of our engineered biosensors have been applied todetect the presence and activity of elastase, an enzyme released by the cercarial larvae stageof Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes areresponsible for the infection of an estimated 200 million people worldwide. Since ourbiosensors are maintained in lyophilised cells, they could be applied for the detection of S.mansoni and other parasites in settings without reliable cold chain access.
Storch M, Casini A, Mackrow B, et al., 2015, BASIC: a new Biopart Assembly Standard for Idempotent Cloning provides accurate, single-tier DNA assembly for synthetic biology, ACS Synthetic Biology, Vol: 4, Pages: 781-787, ISSN: 2161-5063
The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organisation, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels.
Casini A, Storch M, Baldwin GS, et al., 2015, Bricks and blueprints: methods and standards for DNA assembly, Nature Reviews Molecular Cell Biology, Vol: 16, Pages: 568-576, ISSN: 1471-0080
DNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade, a plethora of powerful new DNA assembly methods — including Gibson Assembly, Golden Gate and ligase cycling reaction (LCR) — have been developed. In this Innovation article, we discuss these methods as well as standards such as the modular cloning (MoClo) system, GoldenBraid, modular overlap-directed assembly with linkers (MODAL) and PaperClip, which have been developed to facilitate a streamlined assembly workflow, to aid the exchange of material between research groups and to create modular reusable DNA parts.
Synthetic Biology - A Primer (Revised Edition) presents an updated overview of the field of synthetic biology and the foundational concepts on which it is built. This revised edition includes new literature references, working and updated URL links, plus some new figures and text where progress in the field has been made. The book introduces readers to fundamental concepts in molecular biology and engineering and then explores the two major themes for synthetic biology, namely ‘bottom-up’ and ‘top-down’ engineering approaches. ‘Top-down’ engineering uses a conceptual framework of systematic design and engineering principles focused around the Design-Build-Test cycle and mathematical modelling. The ‘bottom-up’ approach involves the design and building of synthetic protocells using basic chemical and biochemical building blocks from scratch exploring the fundamental basis of living systems. Examples of cutting-edge applications designed using synthetic biology principles are presented, including: the production of novel, microbial synthesis of pharmaceuticals and fine chemicals the design and implementation of biosensors to detect infections and environmental waste. The book also describes the Internationally Genetically Engineered Machine (iGEM) competition, which brings together students and young researchers from around the world to carry out summer projects in synthetic biology. Finally, the primer includes a chapter on the ethical, legal and societal issues surrounding synthetic biology, illustrating the integration of social sciences into synthetic biology research. Final year undergraduates, postgraduates and established researchers interested in learning about the interdisciplinary field of synthetic biology will benefit from this up-to-date primer on synthetic biology.
Robinson T, Valluri P, Kennedy G, et al., 2014, Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy, Analytical Chemistry, Vol: 86, Pages: 10732-10740, ISSN: 0003-2700
Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.
Casini A, Christodoulou G, Freemont PS, et al., 2014, R2oDNA Designer: Computational Design of Biologically Neutral Synthetic DNA Sequences, ACS SYNTHETIC BIOLOGY, Vol: 3, Pages: 525-528, ISSN: 2161-5063
Casini A, MacDonald JT, De Jonghe J, et al., 2013, One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy, Nucleic Acids Research, Vol: 42, ISSN: 1362-4962
Overlap-directed DNA assembly methods allowmultiple DNA parts to be assembled together inone reaction. These methods, which rely onsequence homology between the ends of DNAparts, have become widely adopted in syntheticbiology, despite being incompatible with a key principleof engineering: modularity. To answer this, wepresent MODAL: a Modular Overlap-DirectedAssembly with Linkers strategy that brings modularityto overlap-directed methods, allowing assemblyof an initial set of DNA parts into a variety ofarrangements in one-pot reactions. MODAL isaccompanied by a custom software tool thatdesigns overlap linkers to guide assembly,allowing parts to be assembled in any specifiedorder and orientation. The in silico design of syntheticorthogonal overlapping junctions allows formuch greater efficiency in DNA assembly for avariety of different methods compared with usingnon-designed sequence. In tests with three differentassembly technologies, the MODAL strategy givesassembly of both yeast and bacterial plasmids,composed of up to five DNA parts in the kilobaserange with efficiencies of between 75 and 100%.It also seamlessly allows mutagenesis to beperformed on any specified DNA parts duringthe process, allowing the one-step creation of constructlibraries valuable for synthetic biologyapplications.
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