A new plasmid display technology for the in vitro selection of functional phenotype–genotype linked proteins

Background: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype–genotype link between the displayed protein and the encoding gene. Results: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50–DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein–plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype–genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Use of phage display technology to investigate allergen-antibody interactions

Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. It provides valuable data when applied to the investigation of IgE interaction with allergens. The aim of this rostrum article is to provide an explanation of the potential of phage display for increasing the understanding of allergen- IgE interaction, the discovery of diagnostic reagents, and the development of novel therapeutics for the treatment of allergic disease. The significance of initial studies that have applied phage display technology in allergy research will be highlighted. Phage display has been used to clone human IgE to timothy grass pollen allergen Phl p 5, to characterize the epitopes for murine and human antibodies to a birch pollen allergen Bet v 1, and to elucidate the epitopes of a murine mAb to the house dust mite allergen Der p 1. The technology has identified peptides that functionally mimic sites of human IgE constant domains and that were used to raise antiserum for blocking binding of IgE to the FcεRI on basophils and subsequent release of histamine. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions. Many advances from these early studies are likely as phage display technology evolves and allergists gain expertise in its research applications.

Solution-processed dual-gate polymer field-effect transistors for display applications

We describe the advantages of dual-gate thin-film transistors (TFTs) for display applications. We show that in TFTs with active semiconductor layers composed of diketopyrrolopyrrole-naphthalene copolymer, the on-current is increased, the off-current is reduced, and the sub-threshold swing is improved compared to single-gate devices. Charge transport measurements in steady-state and under non-quasi-static conditions reveal the reasons for this improved performance. We show that in dual-gate devices, a much smaller fraction of charge carriers move in slow trap states. We also compare the activation energies for charge transport in the top-gate and bottom-gate configurations.

Cubic silsesquioxanes for use in solution processable organic light emitting diodes (OLED)

Commercial products using organic light emitting diode (OLED) display technology have begun to appear in cell phones, mp3 players and even televisions. One key area that has allowed and will allow for this technology to continue its ascension into the flat panel display and lighting markets is materials R and D. From this perspective, recent progress in cubic silsesquioxane (SSQ) based materials may provide some new advantageous properties well suited for OLEDs. In this feature article we provide an overview of recent progress in the synthesis, characterization and implementation of SSQ-based materials with properties well suited for application in solution processable organic/polymer electronics, specifically OLEDs.

Raman microscopy of formamide-intercalated kaolinites treated by controlled-rate thermal analysis technology

Raman spectroscopy of formamide-intercalated kaolinites treated using controlled-rate thermal analysis technology (CRTA), allowing the separation of adsorbed formamide from intercalated formamide in formamide-intercalated kaolinites, is reported. The Raman spectra of the CRTA-treated formamide-intercalated kaolinites are significantly different from those of the intercalated kaolinites, which display a combination of both intercalated and adsorbed formamide. An intense band is observed at 3629 cm-1, attributed to the inner surface hydroxyls hydrogen bonded to the formamide. Broad bands are observed at 3600 and 3639 cm-1, assigned to the inner surface hydroxyls, which are hydrogen bonded to the adsorbed water molecules. The hydroxyl-stretching band of the inner hydroxyl is observed at 3621 cm-1 in the Raman spectra of the CRTA-treated formamide-intercalated kaolinites. The results of thermal analysis show that the amount of intercalated formamide between the kaolinite layers is independent of the presence of water. Significant differences are observed in the CO stretching region between the adsorbed and intercalated formamide.

Cases from the technology disciplines

This document is a collection of ‘cases’ adapted from interviews with supervisors of higher degree research students from the technology disciplines. The supervisors come from a wide range of sub disciplines and represent many levels of experience. We follow in this document Hammond and Ryland’s (2009)2 suggested ranking of supervision experience:  No completions – No experience or new supervisors, with no doctoral completions as principal supervisor  Experienced – 1 to 5 doctoral completions as principal supervisor  Very experienced – over 6 doctoral completions as principal supervisor The cases focus attention on thinking about supervision as a teaching and learning practice; a dimension of higher degree research supervision that is increasingly being recognized as important. They are offered as prompts for individuals and groups of supervisors in thinking about their supervision as a teaching and learning practice.

Planning for smart urban ecosystems : information technology applications for capacity building in environmental decision making

Since the industrial revolution, our world has experienced rapid and unplanned industrialization and urbanization. As a result, we have had to cope with serious environmental challenges. In this context, an explanation of how smart urban ecosystems can emerge, gains a crucial importance. Capacity building and community involvement have always been key issues in achieving sustainable development and enhancing urban ecosystems. By considering these, this paper looks at new approaches to increase public awareness of environmental decision making. This paper will discuss the role of Information and Communication Technologies (ICT), particularly Webbased Geographic Information Systems (Web-based GIS) as spatial decision support systems to aid public participatory environmental decision making. The paper also explores the potential and constraints of these webbased tools for collaborative decision making.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Capital music : personal expression with a public display of song choice

Using information and communication technology devices in public urban places can help to create a personalised space. Looking at a mobile phone screen or listening to music on an MP3 player is a common practice avoiding direct contact with others e.g. whilst using public transport. However, such devices can also be utilised to explore how to build new meaningful connections with the urban space and the collocated people within. We present findings of work-in-progress on Capital Music, a mobile application enabling urban dwellers to listen to music songs as usual, but also allowing them to announce song titles and discover songs currently being listened to by other people in the vicinity. We study the ways that this tool can change or even enhance people’s experience of public urban spaces. Our first user study also found changes in choosing different songs. Anonymous social interactions based on users’ music selection are implemented in the first iteration of the prototype that we studied.

High resolution tiled display walls

Scalable high-resolution tiled display walls are becoming increasingly important to decision makers and researchers because high pixel counts in combination with large screen areas facilitate content rich, simultaneous display of computer-generated visualization information and high-definition video data from multiple sources. This tutorial is designed to cater for new users as well as researchers who are currently operating tiled display walls or 'OptiPortals'. We will discuss the current and future applications of display wall technology and explore opportunities for participants to collaborate and contribute in a growing community. Multiple tutorial streams will cover both hands-on practical development, as well as policy and method design for embedding these technologies into the research process. Attendees will be able to gain an understanding of how to get started with developing similar systems themselves, in addition to becoming familiar with typical applications and large-scale visualisation techniques. Presentations in this tutorial will describe current implementations of tiled display walls that highlight the effective usage of screen real-estate with various visualization datasets, including collaborative applications such as visualcasting, classroom learning and video conferencing. A feature presentation for this tutorial will be given by Jurgen Schulze from Calit2 at the University of California, San Diego. Jurgen is an expert in scientific visualization in virtual environments, human-computer interaction, real-time volume rendering, and graphics algorithms on programmable graphics hardware.

A customisable dashboard display for environmental performance visualisations

We conducted an exploratory study of a mobile energy monitoring tool: The Dashboard. Our point of departure from prior work was the emphasis of end-user customisation and social sharing. Applying extensive feedback, we deployed the Dashboard in real-world conditions to socially linked research participants for a period of five weeks. Participants were encouraged to devise, construct, place, and view various data feeds. The aim of our study was to test the assumption that participants, having control over their Dashboard configuration, would engage, and remain engaged, with their energy feedback throughout the trial. Our research points to a set of design issues surrounding the adoption and continued use of such tools. A novel finding of our study is the impact of social links between participants and their continued engagement with the Dashboard. Our results also illustrate the emergence of energy-voyeurism, a form of social energy monitoring by peers.

The Spartan-3E tutorial 3 : using the LCD display [version 1.0]

This tutorial is designed to assist users who wish to use the LCD screen on the Spartan-3E board. In this tutorial, the PicoBlaze microcontroller is used to control the LCD. The tutorial is organised into three Parts. In Part A, code is written to display the message "Hello World" on the LCD. Part B demonstrates how to define and display custom characters. Finally, Part C shows how the display can be shifted and flashed. Shifting is done by using a delay in the main PicoBlaze program loop, while flashing is done using the PicoBlaze interrupt. The slider switches can be used to select the shifting direction, and to turn shifting and flashing on and off.