5,198 research outputs found
A design of experiments approach for the rapid formulation of a chemically defined medium for metabolic profiling of industrially important microbes
This is the final version. Available on open access from Public Library of Science via the DOI in this recordData Availability: All relevant data are within the manuscript and its Supporting Information files.Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have utilised semi-defined media recipes that contain complex, undefined, biologically derived nutrients which have unknown ingredients that cannot be quantified during metabolic profiling. This study used design of experiments to investigate how individual nutrients and interactions between these nutrients contribute to growth. A mathematically derived defined medium has been formulated that has been shown to robustly support growth of G. thermoglucosidans in two different environmental conditions (96-well plate and shake flask) and with a variety of lignocellulose-based carbohydrates. This enabled the catabolism of industrially relevant carbohydrates to be investigated.Shell Global Solutions BVUniversity of Exete
Design of Experiments Methodology to Build a Multifactorial Statistical Model Describing the Metabolic Interactions of Alcohol Dehydrogenase Isozymes in the Ethanol Biosynthetic Pathway of the Yeast Saccharomyces cerevisiae
This is the author accepted manuscript. The final version is available from the American Chemical Society via the DOI in this recordMultifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.We wish to thank Dr. Alex Johns for helpful discussions. S.R.B. would also like to thank Shell Biodomain for funding for this PhD research project
Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications
This is the author accepted manusript. The final version is available from American Chemical Society via the DOI in this recordAccession Codes:
The sequence data for the four Geobacillus spp. used in this study have been submitted to the NCBI Sequence Read Archive and are available under the accession number PRJNA521450.Well-characterized promoter collections for synthetic biology applications are not always available in industrially relevant hosts. We developed a broadly applicable method for promoter identification in atypical microbial hosts that requires no a priori understanding of cis-regulatory element structure. This novel approach combines bioinformatic filtering with rapid empirical characterization to expand the promoter toolkit and uses machine learning to improve the understanding of the relationship between DNA sequence and function. Here, we apply the method in Geobacillus thermoglucosidasius, a thermophilic organism with high potential as a synthetic biology chassis for industrial applications. Bioinformatic screening of G. kaustophilus, G. stearothermophilus, G. thermodenitrificans, and G. thermoglucosidasius resulted in the identification of 636 100 bp putative promoters, encompassing the genome-wide design space and lacking known transcription factor binding sites. Eighty of these sequences were characterized in vivo, and activities covered a 2-log range of predictable expression levels. Seven sequences were shown to function consistently regardless of the downstream coding sequence. Partition modeling identified sequence positions upstream of the canonical -35 and -10 consensus motifs that were predicted to strongly influence regulatory activity in Geobacillus, and artificial neural network and partial least squares regression models were derived to assess if there were a simple, forward, quantitative method for in silico prediction of promoter function. However, the models were insufficiently general to predict pre hoc promoter activity in vivo, most probably as a result of the relatively small size of the training data set compared to the size of the modeled design space
On the elastic moduli of three-dimensional assemblies of spheres: characterization and modeling of fluctuations in the particle displacement and rotation
The elastic moduli of four numerical random isotropic packings of Hertzian
spheres are studied. The four samples are assembled with different preparation
procedures, two of which aim to reproduce experimental compaction by vibration
and lubrication. The mechanical properties of the samples are found to change
with the preparation history, and to depend much more on coordination number
than on density.
Secondly, the fluctuations in the particle displacements from the average
strain are analysed, and the way they affect the macroscopic behavior analyzed.
It is found that only the average over equally oriented contacts of the
relative displacement these fluctuations induce is relevant at the macroscopic
scale. This average depends on coordination number, average geometry of the
contact network and average contact stiffness. As far as the separate
contributions from particle displacements and rotations are concerned, the
former is found to counteract the average strain along the contact normal,
while the latter do in the tangential plane. Conversely, the tangential
components of the center displacements mainly arise to enforce local
equilibrium, and have a small, and generally stiffening effect at the
macro-scale.
Finally, the fluctuations and the shear modulus that result from two
approaches available in the literature are estimated numerically. These
approaches are both based on the equilibrium of a small-sized representative
assembly. The improvement of these estimate with respect to the average strain
assumption indicates that the fluctuations relevant to the macroscopic behavior
occur with short correlation length.Comment: Submitted to IJS
Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli.
This is the final version of the article. Available from National Academy of Sciences via the DOI in this record.Data deposition: The synthetic nucleotide sequences reported in this paper have been deposited in GenBank database (accession nos. JQ901708, JQ901709, and JQ901710).Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.This work was supported by a grant from Shell Research Ltd. and a Biotechnology and Biological Sciences Research Council (BBSRC) Industry Interchange Partnership grant (to J.L.)
The centrosome and spindle as a ribonucleoprotein complex
Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Chromosome Research 19 (2011): 367-376, doi:10.1007/s10577-011-9186-7.The presence of nucleic acids in centrosomes and the spindle have been proposed,
observed, and reported since the 1950s. Why did the subject remain, perhaps even until
today, such a controversial issue? The explanation is manifold, and includes legitimate
concern over contamination from other cellular compartments in biochemical
preparations. With a typically high background of cytoplasmic ribosomes, even
microscopic images of stained intact cells could be difficult to interpret. Also, evidence
for RNA and DNA in centrosomes accumulated for approximately 40 years but was
interspersed with contradictory studies, primarily regarding the presence of DNA
(reviewed in Johnson and Rosenbaum, 1991; Marshall and Rosenbaum, 2000). Perhaps
less tangible but still a likely cause for lingering controversy is that the presence of
nucleic acids in the spindle or centrosomes will require us to look differently at these
structures from a functional, and more to the point, evolutionary standpoint.This work was supported by grants from the NIH (GM088503) and NSF (MCB0843092)
to MCA
Development of a thermal ionizer as ion catcher
An effective ion catcher is an important part of a radioactive beam facility
that is based on in-flight production. The catcher stops fast radioactive
products and emits them as singly charged slow ions. Current ion catchers are
based on stopping in He and H gas. However, with increasing intensity of
the secondary beam the amount of ion-electron pairs created eventually prevents
the electromagnetic extraction of the radioactive ions from the gas cell. In
contrast, such limitations are not present in thermal ionizers used with the
ISOL production technique. Therefore, at least for alkaline and alkaline earth
elements, a thermal ionizer should then be preferred. An important use of the
TRIP facility will be for precision measurements using atom traps. Atom
trapping is particularly possible for alkaline and alkaline earth isotopes. The
facility can produce up to 10 s of various Na isotopes with the
in-flight method. Therefore, we have built and tested a thermal ionizer. An
overview of the operation, design, construction, and commissioning of the
thermal ionizer for TRIP will be presented along with first results for
Na and Na.Comment: 10 pages, 4 figures, XVth International Conference on Electromagnetic
Isotope Separators and Techniques Related to their Applications (EMIS 2007
On-chip two-mode division multiplexing using tapered directional coupler-based mode multiplexer and demultiplexer
Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC): A phase I dose escalating safety study and randomised dose expansion of AZD8931 in combination with oxaliplatin and capecitabine chemotherapy in patients with oesophagogastric adenocarcinoma
Background: AZD8931 has equipotent activity against epidermal growth factor receptor, erbB2, and erbB3. Primary objectives were to determine the recommended phase II dose (RP2D) of AZD8931 + chemotherapy, and subsequently assess safety/preliminary clinical activity in patients with operable oesophagogastric cancer (OGC).
Methods: AZD8931 (20 mg, 40 mg or 60 mg bd) was given with Xelox (oxaliplatin + capecitabine) for eight 21-day cycles, continuously or with intermittent schedule (4 days on/3 off every week; 14 days on/7 off, per cycle) in a rolling-six design. Subsequently, patients with OGC were randomised 2:1 to AZD8931 + Xelox at RP2D or Xelox only for two cycles, followed by radical oesophagogastric surgery. Secondary outcomes were safety, complete resection (R0) rate, six-month progression-free survival (PFS) and overall survival.
Results: During escalation, four dose-limiting toxicities were observed among 24 patients: skin rash (1) and failure to deliver 100% of Xelox because of treatment-associated grade III-IV adverse events (AEs) (3: diarrhoea and vomiting; vomiting; fatigue). Serious adverse events (SAE) occurred in 15 of 24 (63%) patients. RP2D was 20-mg bd with the 4/3 schedule. In the expansion phase, 2 of 20 (10%) patients in the Xelox + AZD8931 group and 5/10 (50%) patients in the Xelox group had grade III–IV AEs. Six-month PFS was 85% (90% CI: 66%–94%) in Xelox + AZD8931 and 100% in Xelox alone. Seven deaths (35%) occurred with Xelox + AZD8931 and one (10%) with Xelox. R0 rate was 45% (9/20) with Xelox + AZD8931 and 90% (9/10) with Xelox-alone (P = 0.024).
Conclusion: Xelox + AZD8931 (20 mg bd 4/3 days) has an acceptable safety profile administered as neoadjuvant therapy in operable patients with OGC. (Trial registration: EudraCT 2011-003169-13, ISRCTN-68093791)
Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii.
Botryococcus braunii is a colonial microalga that appears early in the fossil record and is a sensitive proxy of environmental and hydroclimatic conditions. Palaeozoic Botryococcus fossils which contribute up to 90% of oil shales and approximately 1% of crude oil, co-localise with diagnostic geolipids from the degradation of source-signature hydrocarbons. However more recent Holocene sediments demonstrate no such association. Consequently, Botryococcus are identified in younger sediments by morphology alone, where potential misclassifications could lead to inaccurate paleoenvironmental reconstructions. Here we show that a combination of flow cytometry and ancient DNA (aDNA) sequencing can unambiguously identify Botryococcus microfossils in Holocene sediments with hitherto unparalleled accuracy and rapidity. The application of aDNA sequencing to microfossils offers a far-reaching opportunity for understanding environmental change in the recent geological record. When allied with other high-resolution palaeoenvironmental information such as aDNA sequencing of humans and megafauna, aDNA from microfossils may allow a deeper and more precise understanding of past environments, ecologies and migrations
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