Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits

Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential.

Enantioselective Total Synthesis of Diketopiperazinecontaining Natural Products: (–)-Lansai B, (+)-Nocardioazines A and B, and (–)-Acetylapoaranotin

Diketopiperazine (DKP) motif is found in a wide range of biologically active natural products. This work details our efforts toward two classes of DKP-containing natural products.

Class one features the pyrroloindoline structure, derived from tryptophans. Our group developed a highly enantioselective (3 + 2) formal cycloaddition between indoles and acrylates to provide pyrroloindoline products possessing three stereocenters. Utilizing this methodology, we accomplished asymmetric total synthesis of three natural products: (–)-lansai B, (+)-nocardioazines A and B. Total synthesis of (–)-lansai B was realized in six steps, and featured an amino acid dimerization strategy. The total synthesis of (+)-nocardioazine B was also successfully completed in ten steps. Challenges were met in approaching (+)-nocardioazine A, where a seemingly easy last-step epoxidization did not prove successful. After re-examining our synthetic strategy, an early-stage epoxidation strategy was pursued, which eventually yielded a nine-step total synthesis of (+)-nocardioazine A.

Class two is the epidithiodiketopiperazine (ETP) natural products, which possesses an additional episulfide bridge in the DKP core. With the goal of accessing ETPs with different peripheral structures for structure-activity relationship studies, a highly divergent route was successfully developed, which was showcased in the formal synthesis of (–)-emethallicin E and (–)-haematocin, and the first asymmetric synthesis of (–)-acetylapoaranotin.

Ratio of natural isotopes of nitrogen. I. Primary results: soils of Dombes [Translation from: Revue d'Ecologie et de Biologie du Sol 14, 279-287, 1977]

Utilization of the heavy isotope of nitrogen as a tracer has found numerous applications in soil biology. It allows better definition of different stages of the nitrogen cycle, in particular the immobilization-mineralization cycle. In this work, the authors report the results of calculations of natural isotope ratios of nitrogen in samples of water, soil and vegetation prevailing in Dombes and discuss the possibilities of errors and coefficients of fractionation.

Avaliação de propriedades de artefatos à base de borracha natural

A combinação de excelentes propriedades mecânicas aliadas à biocompatibilidade torna a borracha natural um material amplamente empregado na fabricação de artefatos voltados à saúde, tais como bicos de chupetas e de mamadeiras, e luvas cirúrgicas e de procedimentos não-cirúrgicos. Esses artefatos são processados a partir de látices de poli(cis-isopreno) extraído de seringueiras. Entretanto, o grande número de insaturações presentes na cadeia polimérica, mesmo após a vulcanização, resulta em uma baixa resistência desse material ao envelhecimento, causado principalmente pela associação de fatores como temperatura, oxigênio, ozônio e radiação. Outro inconveniente associado à aplicação da borracha natural é a presença de proteínas no látex, com potencial alergênico, podendo promover reações fisiológicas em alguns usuários. Por esse motivo artefatos comerciais à base de material sintético também são encontrados no mercado. Esses artefatos também não são isentos dos processos naturais de desgaste e decomposição. Esta Dissertação teve como objetivo realizar um estudo comparativo de propriedades dos produtos comerciais à base de borracha natural e à base de material sintético (bicos de chupeta e de mamadeiras e luvas) de diferentes fabricantes, como adquiridos e após sofrerem envelhecimento acelerado. O estudo foi feito empregando-se as técnicas de espectroscopia na região do infravermelho com transformada de Fourier (FTIR) e termogravimetria (TG). Uma avaliação mecânica com métodos padronizados foi também realizada

Longitudinal dispersion in laboratory and natural streams

This study concerns the longitudinal dispersion of fluid particles which are initially distributed uninformly over one cross section of a uniform, steady, turbulent open channel flow. The primary focus is on developing a method to predict the rate of dispersion in a natural stream.

Taylor's method of determining a dispersion coefficient, previously applied to flow in pipes and two-dimensional open channels, is extended to a class of three-dimensional flows which have large width-to-depth ratios, and in which the velocity varies continuously with lateral cross-sectional position. Most natural streams are included. The dispersion coefficient for a natural stream may be predicted from measurements of the channel cross-sectional geometry, the cross-sectional distribution of velocity, and the overall channel shear velocity. Tracer experiments are not required.

Large values of the dimensionless dispersion coefficient D/rU* are explained by lateral variations in downstream velocity. In effect, the characteristic length of the cross section is shown to be proportional to the width, rather than the hydraulic radius. The dimensionless dispersion coefficient depends approximately on the square of the width to depth ratio.

A numerical program is given which is capable of generating the entire dispersion pattern downstream from an instantaneous point or plane source of pollutant. The program is verified by the theory for two-dimensional flow, and gives results in good agreement with laboratory and field experiments.

Both laboratory and field experiments are described. Twenty-one laboratory experiments were conducted: thirteen in two-dimensional flows, over both smooth and roughened bottoms; and eight in three-dimensional flows, formed by adding extreme side roughness to produce lateral velocity variations. Four field experiments were conducted in the Green-Duwamish River, Washington.

Both laboratory and flume experiments prove that in three-dimensional flow the dominant mechanism for dispersion is lateral velocity variation. For instance, in one laboratory experiment the dimensionless dispersion coefficient D/rU* (where r is the hydraulic radius and U* the shear velocity) was increased by a factory of ten by roughening the channel banks. In three-dimensional laboratory flow, D/rU* varied from 190 to 640, a typical range for natural streams. For each experiment, the measured dispersion coefficient agreed with that predicted by the extension of Taylor's analysis within a maximum error of 15%. For the Green-Duwamish River, the average experimentally measured dispersion coefficient was within 5% of the prediction.

Aspects of the washout of salmonid eggs. 3. Observations on the settlement of artifical eggs in a section of natural stream channel at several different discharges

It is of value to know the approximate distance of travel at different stream discharges and/or water velocities, of salmonid eggs which have been displaced from redds by spates. This report describes studies in 20 m of stream channel upstream of the fish trap in Dubby Sike. Observations were made on the relation- ships between discharge and water depth and velocity and also on the relationships between water velocity and the settlement of artificial trout eggs. The main aim was to test the hypothesis that, at any given water velocity, eggs would drift smaller distances in a natural stream than in the experimental channels.

Ecoturismo e educação ambiental no contexto da Baixada Fluminense: um diagnóstico das escolas do entorno do Parque Natural Municipal de Nova Iguaçu-RJ

O processo de construção de uma proposta pedagógica de Educação Ambiental no contexto global e interdisciplinar exige um profundo embasamento teórico e prático vindo de diferentes áreas do conhecimento, inclusive da Geografia. Confirmando essa tendência, propõe-se, aqui, uma análise das ações de Educação Ambiental desenvolvida por cinco escolas da Rede Municipal de Educação de Nova Iguaçu - RJ, situadas na Unidade Regional de Governo Centro, área de influência do Parque Natural Municipal de Nova Iguaçu, no período, entre os anos de 2005 e 2010. O Parque aguça o interesse dos atores sociais à participação. Portanto, centra-se nesta constatação o objetivo desta dissertação: produzir um diagnóstico que possibilite subsidiar estudos e proposições para as escolas do entorno do Parque nas ações de Educação Ambiental e que atenda satisfatoriamente a comunidade escolar de modo que possam organizar, participar, agregar, integrar, futuramente, uma rede de planejamento e monitoramento ecoturístico. As proposições estão alicerçadas nas diretrizes estabelecidas nos Parâmetros Curriculares Nacionais, no Plano de Manejo e no papel das escolas do entorno como difusora de conhecimentos e práticas conservacionistas no meio ambiente local. O diagnóstico realizado através de avaliações investigativas revelou informações importantes que subsidiarão um novo referencial para o tema transversal Meio Ambiente, além de levantar o caráter emergencial da qualificação específica do gestor do Parque, da capacitação contínua do educador e, por conseguinte, da formação do educando apto a interagir com o seu entorno imediato, representado pelo Parque e com as escalas mais amplas que envolvem a percepção do Meio Ambiente.

Normal structures and automorphism groups of t-designs

Combinatorial configurations known as t-designs are studied. These are pairs ˂B, ∏˃, where each element of B is a k-subset of ∏, and each t-design occurs in exactly λ elements of B, for some fixed integers k and λ. A theory of internal structure of t-designs is developed, and it is shown that any t-design can be decomposed in a natural fashion into a sequence of “simple” subdesigns. The theory is quite similar to the analysis of a group with respect to its normal subgroups, quotient groups, and homomorphisms. The analogous concepts of normal subdesigns, quotient designs, and design homomorphisms are all defined and used.

This structure theory is then applied to the class of t-designs whose automorphism groups are transitive on sets of t points. It is shown that if G is a permutation group transitive on sets of t letters and ф is any set of letters, then images of ф under G form a t-design whose parameters may be calculated from the group G. Such groups are discussed, especially for the case t = 2, and the normal structure of such designs is considered. Theorem 2.2.12 gives necessary and sufficient conditions for a t-design to be simple, purely in terms of the automorphism group of the design. Some constructions are given.

Finally, 2-designs with k = 3 and λ = 2 are considered in detail. These designs are first considered in general, with examples illustrating some of the configurations which can arise. Then an attempt is made to classify all such designs with an automorphism group transitive on pairs of points. Many cases are eliminated of reduced to combinations of Steiner triple systems. In the remaining cases, the simple designs are determined to consist of one infinite class and one exceptional case.

Long-term natural acidification of upland sites in Cumbria: Evidence from post-glacial lake sediments

Studies by the Freshwater Biological Association over the last 25 years have supplied data relevant to the levels of acidity in local soils and water before the onset of industrial pollution and current interest in acid rain. This article reviews published analysis from cores of lake sediments, in or near the catchment of the River Duddon. Electron spin resonance spectra of humic acids and iodine values confirm evidence from pollen analysis for a history of progressive acidification of the source material of lake sediments since before 5000 radiocarbon years, in upland catchments of the Lake District. Processes involved included: removal of basic ions from soils by rainfall, the effects of which were intensified by removal by man of deciduous forest; acidification of soils and waters by decomposition products of Calluna and further acidification of waters by Sphagnum species which colonized habitats where drainage became impeded by paludification processes.

Natural habitat and population control mechanism of Crassula helmsii (Australian Swamp Stonecrop) in Australia

The plant Crassula helmsii (Kirk) Cochayne, was likely to become widely distributed and to dominate many damp and wet areas of nature reserves, recreational waters and agricultural drainage of Britain. The aim of this report was to study Australian Swamp Stonecrop in its natural habitat where it is in balance with its environment. This contrasts with its rapid and widespread distribution in the U.K. where its growth interferes with the use of fisheries and amenity lakes but also reduces the value of nature reserves and sites of special scientific interest by suppressing native flora. It was proposed to observe its growth at a variety of sites over its natural distribution and to include some environmental factors, e.g. water-level, water-chemistry (nutrients, acidity and alkalinity), frost-tolerance, salinity, with the help of portable sensors, locally-available services or data. 8 weeks of travel in Australia allowed time to study the plant in its natural habitat including the coastal areas of the southern half of the continent i.e . Western Australia, South Australia, New South Wales, Victoria, Tasmania and southern Queensland. The overall objective was to determine the environmental range by visits to selected sites of Crassula helmsii over its geographic range.

Phase interpretation for polarization-dependent near-field images of high-density gratings

It has been described that the near-field images of a high-density grating at the half self-imaging distance could be different for TE and TM polarization states. We propose that the phases of the diffraction orders play an important role in such polarization dependence. The view is verified through the coincidence of the numerical result of finite-difference time-domain method and the reconstructed results from the rigorous coupled-wave analysis. Field distributions of TE and TM polarizations are given numerically for a grating with period d = 2.3 lambda, which are verified through experiments with the scanning near-field optical microscopy technique. The concept of phase interpretation not only explains the polarization dependence at the half self-imaging distance of gratings with a physical view, but also, it could be widely used to describe the near-field diffraction of a variety of periodic diffractive optical elements whose feature size comparable to the wavelength. (C) 2008 Elsevier B.V. All rights reserved.