Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

A visual environment for the manipulation and integration of JAVA beans

MOTIVATION: Visual programming has the potential to allow non- programmers to redesign and rebuild applications to suit their individual needs. We have built such a visual programming environment, which allows non-programmers to interrogate and combine software components graphically to form new applications. As the needs of the biological community grow, so too will the need for more powerful and easy to use software tools. Intelligent visual programming environments will allow users to design and develop applications easily, so that they can concentrate on the application they wish to build rather than how it is to be done. RESULTS: The environment can read in JAVA Beans, and present relevant information about the beans to the user. The user can then graphically specify how they would like information to flow between the beans by performing simple docking operations. Unnecessary complexities associated with such visual design have been removed by providing intelligent docking of components and visual feedback. With such mechanisms, the complexities of building new applications are reduced. When the biologist has finished the visual construction, the design system is able to generate the new application automatically. The system has been designed specifically to meet the needs of the biological community, and a range of 'BioBeans' are being developed. These include beans for visualization (sequence displays and data visualizers), analysis (feature recognition, error detection) and communication (database access, URL retrieval, DDE communication). AVAILABILITY: Freely available. CONTACT: boyle@synomics.com      

DNA芯片技术研究进展及其在分子营养上的应用

DNA芯片技术是近年发展起来的又一新分子生物学研究工具,可使研究者得以自动化、快速、平行地对大量的生物信息加以分析,在基因组水平上研究基因表达。这种技术为从基因组水平研究基因表达水平与生理反应及生理状况的改变之间的关系提供了强有力的手段。通过比较不同营养水平或不同环境条件下的组织细胞基因达到表达谱差异,可以从基因组水平阐明各种营养成分或环境因素对动物机体的基因表达的影响,从而进一步揭示营养生理的机制和环境对动物影响的机理。DNA芯片技术为分子营养的研究开辟了一条崭新的道路,在从DNA芯片的原理、种类、实验设计、统计方法及在分子营养上的应用作一综述。     

Using computer-aided drug design and medicinal chemistry strategies in the fight against diabetes

The aim of this work is to present a simple, practical and efficient protocol for drug design, in particular Diabetes, which includes selection of the illness, good choice of a target as well as a bioactive ligand and then usage of various computer aided drug design and medicinal chemistry tools to design novel potential drug candidates in different diseases. We have selected the validated target dipeptidyl peptidase IV (DPP-IV), whose inhibition contributes to reduce glucose levels in type 2 diabetes patients. The most active inhibitor with complex X-ray structure reported was initially extracted from the BindingDB database. By using molecular modification strategies widely used in medicinal chemistry, besides current state-of-the-art tools in drug design (including flexible docking, virtual screening, molecular interaction fields, molecular dynamics, ADME and toxicity predictions), we have proposed 4 novel potential DPP-IV inhibitors with drug properties for Diabetes control, which have been supported and validated by all the computational tools used herewith.     

MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions,Dynamics and Formation of Multi-protein Structures

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.     

Molecular epidemiology: a multidisciplinary approach to understanding parasitic zoonoses

Sound application of molecular epidemiological principles requires working knowledge of both molecular biological and epidemiological methods. Molecular tools have become an increasingly important part of studying the epidemiology of infectious agents. Molecular tools have allowed the aetiological agent within a population to be diagnosed with a greater degree of efficiency and accuracy than conventional diagnostic tools. They have increased the understanding of the pathogenicity, virulence, and host-parasite relationships of the aetiological agent, provided information on the genetic structure and taxonomy of the parasite and allowed the zoonotic potential of previously unidentified agents to be determined. This review describes the concept of epidemiology and proper study design, describes the array of currently available molecular biological tools and provides examples of studies that have integrated both disciplines to successfully unravel zoonotic relationships that would otherwise be impossible utilising conventional diagnostic tools. The current limitations of applying these tools, including cautions that need to be addressed during their application are also discussed.     

Express primer tool for high-throughput gene cloning and expression

High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for the simultaneous amplification of multiple target genes.     

RNAsoft: A suite of RNA secondary structure prediction and design software tools

DNA and RNA strands are employed in novel ways in the construction of nanostructures, as molecular tags in libraries of polymers and in therapeutics. New software tools for prediction and design of molecular structure will be needed in these applications. The RNAsoft suite of programs provides tools for predicting the secondary structure of a pair of DNA or RNA molecules, testing that combinatorial tag sets of DNA and RNA molecules have no unwanted secondary structure and designing RNA strands that fold to a given input secondary structure. The tools are based on standard thermodynamic models of RNA secondary structure formation. RNAsoft can be found online at http://www.RNAsoft.ca.     

Utilising structural knowledge in drug design strategies: applications using Relibase

The concept of structure-based drug design is based upon an in-depth understanding of the principles of molecular recognition. Despite our lack of a thorough comprehension of these principles, the wealth of protein structures available opens up unprecedented possibilities for new insights from the analysis of these data. Unravelling universal rules of molecular recognition is certainly one of the most appealing goals. But our knowledge is enhanced also when studying the specific determinants that characterise single targets or target families only, and the factors governing and discriminating their recognition properties.Here, we illustrate how the structure-based design process can benefit from the consequent incorporation of database query tools. We discuss representative examples to address issues such as protein flexibility, water molecules in binding pockets, and ligand specificity as some of the most critical aspects of drug design. All studies are carried out using our database system Relibase. We also show the application of Relibase in searching for preferred geometrical patterns between interacting molecular fragments.     

Cactus Tools for Grid Applications

Cactus is an open source problem solving environment designed for scientists and engineers. Its modular structure facilitates parallel computation across different architectures and collaborative code development between different groups. The Cactus Code originated in the academic research community, where it has been developed and used over many years by a large international collaboration of physicists and computational scientists. We discuss here how the intensive computing requirements of physics applications now using the Cactus Code encourage the use of distributed and metacomputing, and detail how its design makes it an ideal application test-bed for Grid computing. We describe the development of tools, and the experiments which have already been performed in a Grid environment with Cactus, including distributed simulations, remote monitoring and steering, and data handling and visualization. Finally, we discuss how Grid portals, such as those already developed for Cactus, will open the door to global computing resources for scientific users.     

高通量筛选工具的设计与应用

定向进化方法作为新兴的高效蛋白质工程手段,其内容包括蛋白质突变体文库的构建和有效突变体的快速筛选。高通量筛选方法是定向进化方法的重要组成部分,是成功获得有效突变体的关键。筛选的突变体数量越多,获得有效突变体的几率越大。以下介绍了目前已经成功应用于或有潜力应用于定向进化改造蛋白质的几种高通量筛选工具。高通量筛选工具的不断设计与开发将推动蛋白质工程领域的技术革新。     

An affordable approach to interactive desktop molecular modeling

The amazing revolution in computer hardware performance and cost reduction has yet to be carried over to computer software. In fact, application software today is often more expensive and less reliable than the hardware. New enhancements in software development techniques, such as object oriented programming and interactive graphics based user interface design, finally may be having a significant impact on the time-to-market and reliability of these application programs. We discuss our experiences using one such set of software development tools available on the NeXT workstation and describe the effort required to port our MidasPlus molecular modeling package to the NeXT workstation.     

E-photosynthesis: a comprehensive modeling approach to understand chlorophyll fluorescence transients and other complex dynamic features of photosynthesis in fluctuating light

Plants are exposed to a temporally and spatially heterogeneous environment, and photosynthesis is well adapted to these fluctuations. Understanding of the complex, non-linear dynamics of photosynthesis in fluctuating light requires novel-modeling approaches that involve not only the primary light and dark biochemical reactions, but also networks of regulatory interactions. This requirement exceeds the capacity of the existing molecular models that are typically reduced to describe a partial process, dynamics of a specific complex or its particular dynamic feature. We propose a concept of comprehensive model that would represent an internally consistent, integral framework combining information on the reduced models that led to its construction. This review explores approaches and tools that exist in engineering, mathematics, and in other domains of biology that can be used to develop a comprehensive model of photosynthesis. Equally important, we investigated techniques by which one can rigorously reduce such a comprehensive model to models of low dimensionality, which preserve dynamic features of interest and, thus, contribute to a better understanding of photosynthesis under natural and thus fluctuating conditions. The web-based platform www.e-photosynthesis.org is introduced as an arena where these concepts and tools are being introduced and tested.     

Phenotyping for Abiotic Stress Tolerance in Maize

The ability to quickly develop germplasm having tolerance to several complex polygenic inherited abiotic and biotic stresses combined is critical to the resilience of cropping systems in the face of climate change.Molecular breeding offers the tools to accelerate cereal breeding;however,suitable phenotyping proto-cols are essential to ensure that the much-anticipated benefits of molecular breeding can be realized.To facilitate the full potential of molecular tools,greater emphasis needs to be given to reducing the within-experimental site variability,application of stress and characterization of the environment and appropriate phenotyping tools.Yield is a function of many processes throughout the plantcycle,and thus integrative traits that encompass crop performance over time or organization level(i.e.canopy level) will provide a better alternative to instantaneous measurements which provide only a snapshot of a given plant process.Many new phenotyping tools based on remote sensing are now available including non-destructive measurements of growth-related parameters based on spectral reflectance and infrared thermometry to estimate plant water status.Here we describe key field phenotyping protocols for maize with emphasis on tolerance to drought and low nitrogen.     

Vector NTI, a balanced all-in-one sequence analysis suite

Vector NTI is a well-balanced desktop application integrated for molecular sequence analysis and biological data management. It has a centralised database and five application modules: Vector NTI, AlignX, BioAnnotator, ContigExpress and GenomBench. In this review, the features and functions available in this software are examined. These include database management, primer design, virtual cloning, alignments, sequence assembly, 3D molecular viewer and internet tools. Some problems encountered when using this software are also discussed. It is hoped that this review will introduce this software to more molecular biologists so they can make better-informed decisions when choosing computational tools to facilitate their everyday laboratory work. This tool can save time and enhance analysis but it requires some learning on the user's part and there are some issues that need to be addressed by the developer.     

Energy landscapes of rotary DNA origami devices determined by fluorescence particle tracking

Biomolecular nanomechanical devices are of great interest as tools for the processing and manipulation of molecules, thereby mimicking the function of nature’s enzymes. DNA nanotechnology provides the capability to build molecular analogs of mechanical machine elements such as joints and hinges via sequence-programmable self-assembly, which are otherwise known from traditional mechanical engineering. Relative to their size, these molecular machine elements typically do not reach the same relative precision and reproducibility that we know from their macroscopic counterparts; however, as they are scaled down to molecular sizes, physical effects typically not considered by mechanical engineers such as Brownian motion, intramolecular forces, and the molecular roughness of the devices begin to dominate their behavior. In order to investigate the effect of different design choices on the roughness of the mechanical energy landscapes of DNA nanodevices in greater detail, we here study an exemplary DNA origami-based structure, a modularly designed rotor-stator arrangement, which resembles a rotatable nanorobotic arm. Using fluorescence tracking microscopy, we follow the motion of individual rotors and record their corresponding energy landscapes. We then utilize the modular construction of the device to exchange its constituent parts individually and systematically test the effect of different design variants on the movement patterns. This allows us to identify the design parameters that most strongly affect the shape of the energy landscapes of the systems. Taking into account these insights, we are able to create devices with significantly flatter energy landscapes, which translates to mechanical nanodevices with improved performance and behaviors more closely resembling those of their macroscopic counterparts.