Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.     

Microbial xylanases: Engineering, production and industrial applications

Enzymatic depolymerization of hemicellulose to monomer sugars needs the synergistic action of multiple enzymes, among them endo-xylanases (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37) (collectively xylanases) play a vital role in depolymerizing xylan, the major component of hemicellulose. Recent developments in recombinant protein engineering have paved the way for engineering and expressing xylanases in both heterologous and homologous hosts. Functional expression of endo-xylanases has been successful in many hosts including bacteria, yeasts, fungi and plants with yeasts being the most promising expression systems. Functional expression of β-xylosidases is more challenging possibly due to their more complicated structures. The structures of endo-xylanases of glycoside hydrolase families 10 and 11 have been well elucidated. Family F/10 endo-xylanases are composed of a cellulose-binding domain and a catalytic domain connected by a linker peptide with a (β/α)(8) fold TIM barrel. Family G/11 endo-xylanases have a β-jelly roll structure and are thought to be able to pass through the pores of hemicellulose network owing to their smaller molecular sizes. The structure of a β-d-xylosidase belonging to family 39 glycoside hydrolase has been elucidated as a tetramer with each monomer being composed of three distinct regions: a catalytic domain of the canonical (β/α)(8) - TIM barrel fold, a β-sandwich domain and a small α-helical domain with the enzyme active site that binds to d-xylooligomers being present on the upper side of the barrel. Glycosylation is generally considered as one of the most important post-translational modifications of xylanases, but a few examples showed functional expression of eukaryotic xylanases in bacteria. The optimal ratio of these synergistic enzymes is very important in improving hydrolysis efficiency and reducing enzyme dosage but has hardly been addressed in literature. Xylanases have been used in traditional fields such as food, feed and paper industries for a longer time but more and more attention has been paid to using them in producing sugars and other chemicals from lignocelluloses in recent years. Mining new genes from nature, rational engineering of known genes and directed evolution of these genes are required to get tailor-made xylanases for various industrial applications.     

Plant-derived smoke: Old technology with possibilities for economic applications in agriculture and horticulture

Fire and smoke have been used in traditional agricultural systems for centuries. In recent years, biologically active compounds have been isolated from smoke with potential uses in agriculture and horticulture. This article highlights the possibilities of using smoke-water or smoke-derived butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one, termed karrikinolide, KAR₁) for the cultivation of agricultural and horticultural crops. Treatments with smoke-water show promising results for improving seed germination, seedling growth and crop productivity. In certain cases, even under adverse conditions, such as low or high temperatures and low osmotic potentials, smoke-water or a KAR₁ solution can promote seed germination and seedling growth. This phenomenon is of great significance when seeds are sown under drought conditions. Smoke-technology, therefore, has potential for use in arid and semi-arid regions. Possibilities may also exist for controlling some plant diseases and managing weeds with the use of smoke or KAR₁ solutions. In addition, smoke-technology can possibly economize the use of commercial chemical fertilizers, pesticides and herbicides, making it a feasible technology for organic farming and for resource-poor farmers in developing nations. The positive role of smoke-water in flowering and fruiting of crops cannot be overlooked as the karrikins found in smoke are now recognized as potential new plant growth regulators. Very low concentrations of smoke-water or a KAR₁ solution are effective in promoting germination and post-germination growth. Thus, early harvesting and increasing the productivity of crops using smoke-technology may be possible. Here we review some of the effects of smoke and KAR₁ on various crop species and discuss the potential uses of smoke technology in agriculture and horticulture.     

Engineering and applications of genetic circuits

In the field of synthetic biology, recent genetic engineering efforts have enabled the construction of novel genetic circuits with diverse functionalities and unique activation mechanisms. Because of these advances, artificial genetic networks are becoming increasingly complex, and are demonstrating more robust behaviors with reduced crosstalk between defined modules. These properties have allowed for the identification of a growing set of design principles that govern genetic networks, and led to an increased number of applications for genetic circuits in the fields of metabolic engineering and biomedical engineering. Such progress indicates that synthetic biology is rapidly evolving into an integrated engineering practice that uses rational and combinatorial design of synthetic gene networks to solve complex problems in biology, medicine, and human health.     

A resource query interface for network-aware applications

Networked systems provide a cost-effective platform for parallel computing, but the applications have to deal with the changing availability of computation and communication resources. Network-awareness is a recent attempt to bridge the gap between the realities of networks and the demands of applications. Network-aware applications obtain information about their execution environment and dynamically adapt to enhance their performance. Adaptation is especially important for synchronous parallel applications because a single busy communication link can become the bottleneck and degrade overall performance dramatically. This paper presents Remos, a uniform API that allows applications to obtain relevant network information, and reports on the development of parallel applications in this environment. The challenges in defining a uniform interface include network heterogeneity, diversity and variability in network traffic, and resource sharing in the network and even inside an application. The first implementation of the Remos interface uses SNMP to monitor IP-based networks. This paper reports on our methodology for developing adaptive parallel applications for high-speed networks with Remos and presents experimental results using applications generated by the Fx parallelizing compiler. The results highlight the importance and effectiveness of adaptive parallel computing. This revised version was published online in July 2006 with corrections to the Cover Date.     

An integrative method for evaluating the biological effects of nanoparticle-protein corona

BackgroundNanoplastics in the environment can enter the human body through gastrointestinal intake, dermal contact, and pulmonary inhalation, posing a threat to human health. Protein molecules in body fluids will quickly adsorb on the surfaces of the nanoplastics, forming a protein corona, which has implications for the interaction of the nanoplastics with cells and the metabolic pathways of the nanoplastic within cells. For years, practical tools such as dynamic light scattering, transmission electron microscopy, and liquid chromatography have been developed to understand the protein corona of nanoparticles (NPs), either in vitro or in cellular or molecular level. However, an integrated approach to understand the nanoparticles-protein corona is still lacking.MethodsUsing the most frequently observed environmental nanoplastics, polystyrene nanoplastics (PS), as a standard, we established an integrative structural characterization platform, a biophysical and biochemical evaluation method to investigate the effect of surface charge on protein corona composition. The cellular and molecular mechanisms were also explored through in vitro cellular experiments.ResultsThe first integrative method for characterizing biological properties of NPs-protein corona has been established. This method comprehensively covers the critical aspects to understand NPs-protein corona interactions, from structure to function.ConclusionsThe integrative method for nanoplastics microstructure characterization can be applied to the structural characterization of nanoparticles in nanoscale, which is of universal significance from in vitro characterization to cellular experiments and then to molecular mechanism studies.General significanceThis strategy has high reliability and repeatability and can be applied both in environment and nanomedicine safety assessment.     

Engineering antibodies for clinical applications

Molecular engineering has contributed immensely to the clinical success of antibodies in recent years. The modular structure of antibodies has permitted their modification in numerous ways, to meet various clinical requirements. With the help of antibody engineering, it has been possible to modify the molecular size, pharmacokinetics, immunogenicity, binding affinity, specificity and effector function of antibodies. In addition, fusion proteins of antibodies with various proteins and peptides have yielded targeted biological modifiers, toxins and imaging agents. This review focuses on the recent trends in antibody engineering for improving their clinical utility.     

Engineering blood vessels from stem cells: recent advances and applications

Endothelial cells organized into blood vessels are critical for the formation and maintenance of most tissues in the body and are involved in regulating physiological processes such as angiogenesis, inflammation and thrombosis. Endothelial cells are of great research interest, because of their potential to treat vascular diseases and to stimulate the growth of ischaemic tissue. They can be used to engineer artificial vessels, repair damaged vessels, and to induce the formation of vessel networks in engineered tissues. For such clinical applications, proliferating human endothelial progenitor cells can be isolated from adult tissues or embryonic stem cells. Recently, these cells were successfully used to engineer single vessels and to stimulate capillary networks, both in vitro and in vivo.     

Atomic Force Microscopy of the fungi-mineral interface: applications in mineral dissolution, weathering and biogeochemistry

The interaction between mycorrhizal fungi and minerals is of fundamental importance in affecting the geochemical carbon cycle and CO(2) concentration in the atmosphere, alongside roles in soil creation and the release of nutrients. The symbiosis between the fungi and the plant, supported by photosynthesis in the host plant, has as one of its key features the interfacial zone where mineral and fungi come into contact. At this interface, the organism exudes a complex mixture of organic acids, chelating molecules, protons, and extracellular polysaccharide. In this review, examples will be given of recent Atomic Force Microscopy experiments to monitor the colonization of phyllosilicate minerals in sterile controlled microcosm environments containing only tree seedlings, mineral chips and mycorrhizal fungi. The surface activity of the colonizing fungal hyphae is extensive and complex. In complementary experiments involving exposure of minerals surfaces to single organic acids, it has been possible to monitor dissolution at the unit cell level and to extract activation energies for specific dissolution processes, for example 49kJmol(-1) for 100mM oxalic acid acting upon a biotite sample. The link between these simpler model experiments and the whole microcosm studies is illustrated partly by observations of fungal-colonized mineral surfaces from microcosms after careful removal of the organism and biolayer. These mineral surfaces give clear indications of basal plane modification and fungal weathering.     

A parallel programming interface for out-of-core cluster applications

Clusters of workstations are a practical approach to parallel computing that provide high performance at a low cost for many scientific and engineering applications. In order to handle problems with increasing data sets, methods supporting parallel out-of-core computations must be investigated. Since writing an out-of-core version of a program is a difficult task and virtual memory systems do not perform well in some cases, we have developed a parallel programming interface and the support library to provide efficient and convenient access to the out-of-core data. This paper focuses on how these components extend the range of problem sizes that can be solved on the cluster of workstations. Execution time of Jacobi iteration when using our interface, virtual memory and PVFS are compared to characterize the performance for various problem sizes, and it is concluded that our new interface significantly increases the sizes of problems that can be efficiently solved. Jianqi Tang received B.Sc. and M.Sc. from Harbin Institute of Technology in 1997 and 1999 respectively, both in computer application. Currently, she is a Ph.D. candidate at the Department of Computer Science and engineering, Harbin Institute of Technology. She has participated in several National research projects. Her research interests include parallel computing, parallel I/O and grid computing. Binxing Fang received M.Sc. in 1984 from Tsinghua University and Ph.D. from Harbin Institute of Technology in 1989, both in computer science. From 1990 to 1993 he was with National University of Defense Technology as a postdoctor. Since 1984, he is a faculty member at the Department of Computer Science and engineering of Harbin Institute of Technology, where he is presently a Professor. He is a Member of the National Information Expert Consultant Group and a Standing Member of the Council of Chinese Society of Communications. His research efforts focus on parallel computing, computer network and information security. Professor Fang has implemented over 30 projects from the state and ministry/province. Mingzeng Hu was born in 1935. He has been with the Department of Computer Science and engineering in Harbin Institute of Technology since 1958, where he is currently a Professor. He was a visiting scholar in the Siemens Company, Germany from 1978 to 1979, a visiting associate professor in Chiba University, Japan from 1984 to 1985, and a visiting professor in York University, Canada from 1989 to 1995. He is the Director of the National Key Laboratory of Computer Information Content Security. He is also a Member of 3rd Academic Degree Committee under the State Council of China. Professor Hu’s research interests include high performance computer architecture and parallel processing technology, fault tolerant computing, network system, VL design, and computer system security technology. He has implemented many projects from the state and ministry/province and has won several Ministry Science and Technology Progress Awards. He published over 100 papers in core journals home and abroad and one book. Professor Hu has supervised over 20 doctoral students. Hongli Zhang received M.Sc in computer system software in 1996 and Ph.D. in computer architecture in 1999 from Harbin Institute of Technology. Currently, she is an Associate Professor at the Department of Computer Science and engineering, Harbin Institute of Technology. Her research interests include computer network security and parallel computing.     

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering.     

Engineering tubulin: microtubule functionalization approaches for nanoscale device applications

With the emergences of engineered devices at microscale and nanoscale dimensions, there is a growing need for controlled actuation and transport at these length scales. The kinesin–microtubule system provides a highly evolved biological transport system well suited for these tasks. Accordingly, there is an ongoing effort to create hybrid nanodevices that integrate biological components with engineered materials for applications such as biological separations, nanoscale assembly, and sensing. Adopting microtubules for these applications generally requires covalent attachment of biotin, fluorophores, or other biomolecules to tubulin enable surface or cargo attachment, or visualization. This review summarizes different strategies for functionalizing microtubules for application-focused as well as basic biological research. These functionalization strategies must maintain the integrity of microtubule proteins so that they do not depolymerize and can be transported by kinesin motors, while adding utility such as the ability to reversibly bind cargo. The relevant biochemical and electrical properties of microtubules are discussed, as well as strategies for microtubule stabilization and long-term storage. Next, attachment strategies, such as antibodies and DNA hybridization that have proven useful to date, are discussed in the context of ongoing hybrid nanodevice research. The review concludes with a discussion of less explored opportunities, such as harnessing the utility of tubulin posttranslational modifications and the use of recombinant tubulin that may enable future progress in nanodevice development.     

Fermentation scale-up: Problems and possibilities

Existing fermentation scale-up methods cannot meet adequately the technical and economic demands of the modern biotechnology industry. Shortcomings of available methods and potential routes to better ones are discussed in this article.     

MicroRNA sponges: Progress and possibilities

The microRNA (miRNA) “sponge” method was introduced three years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge''s binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a useful tool to probe miRNA functions in a variety of experimental systems. Here we will discuss the ways sponge and related constructs can be optimized and review recent applications of this method with particular emphasis on stable expression in cancer studies and in transgenic animals.     

Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications

Chromosomal rearrangements are important resources for genetic studies. Recently, a Cre-loxP-based method to introduce defined chromosomal rearrangements (deletions, duplications, and inversions) into the mouse genome (chromosome engineering) has been established. To explore the limits of this technology systematically, we have evaluated this strategy on mouse chromosome 11. Although the efficiency of Cre-loxP-mediated recombination decreases with increasing genetic distance when the two endpoints are on the same chromosome, the efficiency is not limiting even when the genetic distance is maximized. Rearrangements encompassing up to three quarters of chromosome 11 have been constructed in mouse embryonic stem (ES) cells. While larger deletions may lead to ES cell lethality, smaller deletions can be produced very efficiently both in ES cells and in vivo in a tissue- or cell-type-specific manner. We conclude that any chromosomal rearrangement can be made in ES cells with the Cre-loxP strategy provided that it does not affect cell viability. In vivo chromosome engineering can be potentially used to achieve somatic losses of heterozygosity in creating mouse models of human cancers.     

The mosquito genome: perspectives and possibilities

Anopheles gambiae is the mosquito vector responsible for transmitting Plasmodium falciparum, a malaria parasite of humans. With the emergence of genome projects for a variety of prokaryotic and eukaryotic microorganisms, there has been a long-standing interest in sequencing the genomes of the malaria parasite and its insect vector. This tour de force effort has now been completed and reported. The alignment of putative orthologs in An. gambiae with those of Drosophila melanogaster highlights several similarities and differences. These findings could have implications in: (1) identifying new targets for insecticide development; (2) strengthening our understanding of the developmental biology of mosquitoes; and (3) possibly controlling pathogen transmission. A brief overview of these interesting findings and the implications for further studies will be discussed here.     

Charting plant interactomes: possibilities and challenges

Protein-protein interactions are essential for nearly all cellular processes. Therefore, an important goal of post-genomic research for defining gene function and understanding the function of macromolecular complexes involves creating 'interactome' maps from empirical or inferred datasets. Systematic efforts to conduct high-throughput surveys of protein-protein interactions in plants are needed to chart the complex and dynamic interaction networks that occur throughout plant development. However, no single approach can build a complete map of the interactome. Here, we review the utility and potential of various experimental approaches for creating large-scale protein-protein interaction maps in plants. Bioinformatics approaches for curating and assessing the confidence of these datasets through inter-species comparisons will be crucial in achieving a complete understanding of protein interaction networks in plants.