C = Coordinator P = Principal contractor

(0)     Coordinator
Dr Keith Firman + University of Portsmouth Finance Department. 
The funds requested will allow us to employ a part-time Coordinator  to assist Dr Firman with day to day administrative tasks involved in coordinating the project.

(1)     Dr. Keith Firman, Portsmouth Group
Science Coordinator, whose research team will prepare and assay activity of the molecular motor (EcoR124I) and provide much of the DNA substrate(s) required for the project.  The Biophysics Laboratories at Portsmouth University (recently rated as a Grade 5 resource in the UK Research Assessment Exercise) have a well-equipped Molecular Biology Facility, which will allow protein preparation, DNA preparation and analysis of the biochemical properties of the motor.  In addition, KF is Network Coordinator for the NanoNet Nanotechnology Network, whose Portsmouth members that are active in this research project, are - Dr Sheelagh Campbell - Will lead the work on the use of the AFM at Portsmouth, with Dr. James Smith.  This AFM work will initially involve confirmation that material such as motor proteins and specific DNA substrates interact as expected.  However, the work will develop toward chemical derivatisation of tips to enable a wide variety of the techniques in the project.  Dr. Darren Mernagh - Will work with Dr. Roberto Favilla (see below) to prepare and analyse fluorescently labelled DNA for studies with FRET signals during translocation and will be closely involved in initial screening of substrates for FRET activity.  Dr David Franklin – Who will be involved, with NPL, in the preparation of magnetic beads for attachment to DNA particularly at the early stage of producing sub-micron magnetic particles to replace commercial beads in the early stages of the work.

(2)     Dr. John Gallop and Dr. Peter Cumpson, The National Physical Laboratory
(NPL) has a world-leading capability in accurate force measurement calibration of scanned probe microscopes.  Work is being carried out on a number of novel methods for calibrating cantilever force constants using photonic methods which may have an important role to play in quantifying, in a traceable way, the working force and mechanical efficiency of the motor.  NPL has a number of scanned probe microscope facilities, which might be used to determine accurately the size, and shape of the magnetic particles based on known reference materials or metrological assessment of size distributions.  This group will be closely involved in both the preparation of nano-sized magnetic particles and also MFM and SQUID assays of their movement.

(3)    Prof. Cees Dekker, Delft University of Technology
Has built its reputation with leading work on the physical properties of carbon nanotubes. Recently the focus has shifted towards exploratory work in single-molecule biophysics, and the group employs AFM, STM, magnetic tweezers, and nanofabricated structures to study biomolecular systems and foster new Nanotechnology. Current research efforts include [1] Studies of the electronic properties of carbon nanotubes and development of nanotube-based scanning probe tips and biosensors. [2] Local probe studies of DNA repair, redox and restriction enzymes, and membrane proteins. [3] Translocation of DNA and proteins through membrane pores and nano-fluidic channels. [4] DNA-mediated assembly of hybrid nanostructures such as nanotubes, C60, and clusters. The group is a part of the Delft Institute for MicroElectronics and Submicron technology (DIMES), the Dutch national facility for device fabrication and nano-structuring. The technical infrastructure is dedicated towards nanofabricated samples made by electron-beam lithography using an EBPG-5 pattern generator. Various cryostats are available as well as home-built ultra-low-noise electronics for sensitive experiments. Recently the group built a magnetic tweezer set-up that allows sensitive force spectroscopy on single biomolecules, in combination with control of the supercoiling state of DNA. A variety of scanning probe microscopes (STM, AFM) is available for the project as well. We will contribute to this project in the following areas - Magnetic tweezers studies of all the forces, stalling, torque involved in DNA translocation by the molecular motor.  Nano-lithography of small (down to 30 nm or so) structures in and on silicon wafers by electron-beam lithography.  Placement of the molecular motor molecules on nanofabricated silicon-based structures.

(4)     Dr. David Bensimon, CNRS/ENS, Paris 
Have developed a unique magnetic trap technique (Strick et al., Science 271,1835 (1996)) to twist and stretch DNA. This has allowed the study of the elastic properties of DNA and to discover a new phase of DNA under high twist (Allemand et al., PNAS 95,14152 (1998)). Since then they have used that set-up to study the interaction of DNA with various enzymes: topoisomerases (Strick et al., Nature 404, 901 (2000)), DNA-polymerases (Maier et al., PNAS 97,12002 (2000)), helicases, transcription factors, type I restriction enzymes, etc. In those studies, they were focusing on the detailed mechanism of action of the enzyme: its step-size, individual rate with and without load, efficiency, etc. These studies give access to a molecular behaviour that is often buried in the inherent averaging of bulk measurements.  We propose to use this set-up to study the activity of type I restriction enzymes and other DNA enzymes and to couple these manipulation measurements with the visualisation of the enzymes using single molecule fluorescent techniques.

(5)     Prof. Roberto Favilla, Dept. of Biochemistry and Molecular Biology and Dept. of Physics, University of Parma, Italy 
The laboratory of Biophysics, is mainly a laboratory of biomolecular spectroscopy. Beyond other optical spectroscopy instrumentation, we have one spectrofluorometer (Perkin-Elmer) to detect stationary fluorescence signals, and one fluorescence lifetime set-up (based on the single photon counting technique). In addition, we have one modern stopped flow apparatus (Bio-Logic), equipped to detect, among others, fluorescence signals. Therefore, we can detect both stationary and time-dependent FRET above the millisecond time scale, on samples composed by many molecules. In order to detect fluorescence at a single molecule level SNOM instrumentation (Department of Physics), AFM facilities (both departments) as well as relative expertise is available. It is our intention to exploit part of the financial support deriving from the project MOL SWITCH, to buy a modular microspectrofluorometer, capable to detect FRET signals from labelled DNA single molecules. This will allow us to carry out the most sophisticated part of the project concerned with the long-term realisation of a single molecule DNA sequencing nanodevice. Finally, at the Department of Biochemistry and Molecular Biology, most of the facilities typical of a modern biochemical laboratory, including single crystal microspectrophotometer, FPLC, HPLC, etc. are available.

(6)   Dr. Marie Weiserova Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
Our group (two senior scientists and number of undergraduate and doctoral students) belongs to the Laboratory of Molecular Genetics of Bacteria. We have good experience in biochemical and genetical analysis of the structure-function of type I restriction modification enzymes as well as with bacterial membrane vesicles and liposomes. Recently we have focused on analysis of mutants of Type I restriction-modification enzymes. Comparison of in vivo detected R-M phenotype with data from in vitro biochemical analysis and sequencing of the mutants help us to identify the important residues involved in protein-protein interaction and DNA-binding. In order to acquire better understanding of the mechanisms of the assembly and function of multimeric complex restriction-modification enzymes type I, the effect of concentration of individual subunits on the function of the system is studied by 2D-protein electrophoresis and computer assisted image analysis. We have developed a very effective procedure involving NEPHGE with the BioRad Mini-Protean II, which gives us the unique chance to visualise EcoKI and EcoR124I systems in their complexity.  We will be mainly involved in mutagenesis of nuclease domain (Motif X) of HsdR subunits. For sequence analysis of collection of mutants the automatic sequencing facility (Vistra DNA sequencer 725) available in the Institute of Microbiology will be used. We might contribute with some sophisticated technologies such as high-resolution 2D- electrophoresis of proteins using Oxford Glycosystems 2D-electrophoresis unit for high-resolution 2D- electrophoresis of proteins and tRNAs with PDQEST gel image acquisition and analysis system.