49 results found
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
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.
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.
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
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.
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.
Cehovin A, Simpson PJ, McDowell MA, et al., 2013, Specific DNA recognition mediated by a type IV pilin, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 3065-3070, ISSN: 0027-8424
Lu D, Silhan J, MacDonald JT, et al., 2012, Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 16852-16857, ISSN: 0027-8424
Kitney RI, 2012, Synthetic Biology - A Primer, Publisher: Imperial College Press London
Silhan J, Nagorska K, Zhao Q, et al., 2012, Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis, Nucleic Acids Research, Vol: 40, Pages: 2065-2075, ISSN: 1362-4962
We have previously demonstrated that the twoExonuclease III (Xth) family members presentwithin the obligate human pathogen Neisseriameningitidis, NApe and NExo, are important forsurvival under conditions of oxidative stress. Ofthese, only NApe possesses AP endonucleaseactivity, while the primary function of NExoremained unclear. We now reveal further functionalspecialization at the level of 30-PO4 processing forNExo. We demonstrate that the bi-functional meningococcalglycosylases Nth and MutM can performstrand incisions at abasic sites in addition to NApe.However, no such functional redundancy existsfor the 30-phosphatase activity of NExo, and thecytotoxicity of 30-blocking lesions is reflectedin the marked sensitivity of a mutant lackingNExo to oxidative stress, compared to strainsdeficient in other base excision repair enzymes. Ahistidine residue within NExo that is responsiblefor its lack of AP endonuclease activity isalso important for its 30-phosphatase activity,demonstrating an evolutionary trade off in enzymefunction at the single amino acid level. This specializationof two Xth enzymes for the 30-end processingand strand-incision reactions has notpreviously been observed and provides a newparadigm within the prokaryotic world for separationof these critical functions during baseexcision repair.
Nagorska K, Silhan J, Li Y, et al., 2012, A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis, MOLECULAR MICROBIOLOGY, Vol: 83, Pages: 1064-1079, ISSN: 0950-382X
Sheppard C, Camara B, Shadrin A, et al., 2011, Inhibition of Escherichia coli RNAp by T7 Gp2 Protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2, JOURNAL OF MOLECULAR BIOLOGY, Vol: 407, Pages: 623-632, ISSN: 0022-2836
Grippon S, Zhao Q, Robinson T, et al., 2011, Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway, Nucleic Acids Research, Vol: 39, Pages: 2593-2603, ISSN: 1362-4962
Mismatch uracil DNA glycosylase (Mug) fromEscherichia coli is an initiating enzyme in thebase-excision repair pathway. As with other DNAglycosylases, the abasic product is potentiallymore harmful than the initial lesion. Since Mug isknown to bind its product tightly, inhibitingenzyme turnover, understanding how Mug bindsDNA is of significance when considering how Muginteracts with downstream enzymes in the baseexcisionrepair pathway. We have demonstrateddifferential binding modes of Mug between its substrateand abasic DNA product using both band shiftand fluorescence anisotropy assays. Mug binds itsproduct cooperatively, and a stoichiometric analysisof DNA binding, catalytic activity and saltdependenceindicates that dimer formation is offunctional significance in both catalytic activity andproduct binding. This is the first report ofcooperativity in the uracil DNA glycosylase superfamilyof enzymes, and forms the basis of productinhibition in Mug. It therefore provides a new perspectiveon abasic site protection and the findingsare discussed in the context of downstream lesionprocessing and enzyme communication in the baseexcision repair pathway.
Ellis T, Adie T, Baldwin GS, 2011, DNA assembly for synthetic biology: from parts to pathways and beyond, INTEGRATIVE BIOLOGY, Vol: 3, Pages: 109-118, ISSN: 1757-9694
We report on the fabrication and characterization of a DNA nanopore detector with integrated tunneling electrodes. Functional tunneling devices were identified by tunneling spectroscopy in different solvents and then used in proof-of-principle experiments demonstrating, for the first time, concurrent tunneling detection and ionic current detection of DNA molecules in a nanopore platform. This is an important step toward ultrafast DNA sequencing by tunneling.
Robinson T, Manning HB, Dunsby C, et al., 2010, Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics, Conference on Microfluidics, BioMEMS, and Medical Microsystems VIII, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Ayub M, Ivanov A, Instuli E, et al., 2010, Nanopore/electrode structures for single-molecule biosensing, Electrochimica Acta, Vol: 55, Pages: 8237-8243
Baldwin GS, Brooks NJ, Robson RE, et al., 2009, DNA Double Helices Recognize Mutual Sequence Homology in a Protein Free Environment, JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS, Vol: 26, Pages: 880-880, ISSN: 0739-1102
Robinson T, Schaerli Y, Wootton R, et al., 2009, Removal of background signals from fluorescence thermometry measurements in PDMS microchannels using fluorescence lifetime imaging, LAB ON A CHIP, Vol: 9, Pages: 3437-3441, ISSN: 1473-0197
Robinson T, Valluri P, Manning HB, et al., 2008, Three-dimensional molecular mapping in a microfluidic mixing device using fluorescence lifetime imaging, OPTICS LETTERS, Vol: 33, Pages: 1887-1889, ISSN: 0146-9592
Briggs LC, Baldwin GS, Miyata N, et al., 2008, Analysis of nucleotide binding to p97 reveals the properties of a tandem AAA hexameric ATPase, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 283, Pages: 13745-13752
Baldwin GS, Brooks NJ, Robson RE, et al., 2008, DNA double helices recognize mutual sequence homology in a protein free environment, J. Phys. Chem. B, Vol: 112, Pages: 1060-1064
Di Noia JM, Williams GT, Chan DTY, et al., 2007, Dependence of antibody gene diversification on uracil excision, Journal of Experimental Medicine, Vol: 204, Pages: 3209-3219, ISSN: 1540-9538
Activation-induced deaminase (AID) catalyses deamination of deoxycytidine to deoxyuridinewithin immunoglobulin loci, triggering pathways of antibody diversifi cation that arelargely dependent on uracil-DNA glycosylase (uracil- N -glycolase [UNG]). Surprisinglyeffi cient class switch recombination is restored to ung / B cells through retroviraldelivery of active-site mutants of UNG, stimulating discussion about the need for UNG ’ suracil-excision activity. In this study, however, we fi nd that even with the overexpressionachieved through retroviral delivery, switching is only mediated by UNG mutants thatretain detectable excision activity, with this switching being especially dependent onMSH2. In contrast to their potentiation of switching, low-activity UNGs are relativelyineffective in restoring transversion mutations at C:G pairs during hypermutation, or inrestoring gene conversion in stably transfected DT40 cells. The results indicate that UNGdoes, indeed, act through uracil excision, but suggest that, in the presence of MSH2,effi cient switch recombination requires base excision at only a small proportion of theAID-generated uracils in the S region. Interestingly, enforced expression of thymine-DNAglycosylase (which can excise U from U:G mispairs) does not (unlike enforced UNG orSMUG1 expression) potentiate effi cient switching, which is consistent with a need eitherfor specifi c recruitment of the uracil-excision enzyme or for it to be active on singlestrandedDNA.
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