Award details

Robotic systems for selection purification and structure determination of novel proteins and RNA aptamers

Reference	BB/C511021/1
Principal Investigator / Supervisor	Professor Alan Berry
Co-Investigators / Co-Supervisors	Professor Stephen Baldwin, Professor Peter Henderson, Professor Michael McPherson, Professor Simon Phillips, Professor Sheena Radford, Professor Peter Stockley
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	113,052
Status	Completed
Type	Research Grant
Start date	01/11/2004
End date	31/10/2005
Duration	12 months

Abstract

The proposal seeks funds for a robotic colony picker and associated microtitre plate filler, a TECAN Evo robot for filling crystallisation master blocks and an AKTA Explorer system for automated protein and aptamer preparation. There is an increased use of combinatorial techniques in modern biochemical research and hence a need to produce and handle libraries of biomolecules efficiently and accurately. Such studies necessarily increase the routine, repetitive aspects of the work and we are committed to automating as much of this type of work as possible. This proposal seeks funds to integrate the selection of novel proteins and nucleic acid aptamers, their purification and their crystallisation for 3D structural studies, and remove bottlenecks that have developed in our shift to high-throughput biological research. This move reflects the trend in biological sciences, and is critical if the UK is to remain internationally competitive. A major part of such work involves the picking of bacterial colonies or phage plaques containing the gene encoding the novel protein or aptamer from growth plates into microtitre plates: a key step in the indexing of the library. If carried out manually, this task is tedious and repetitive and seriously limits the number of colonies that can currently be analysed. In addition, errors at this point can have serious consequences for the integrity of the library. The manual picking of colonies (or plaques) is therefore a major bottleneck in current research, and manually picked libraries are usually limited to a few thousand library members. A robotic colony picker hand handle this number of colonies in about 1 hour, thus removing a major hurdle to large library construction, while concomitantly freeing the researcher to carry out more demanding tasks. We will use the requested colony picker in a range of projects ranging from structure function studies, through directed evolution of new enzyme activities, protein folding studies to optimising structural genomics projects for the improved expression of soluble and membrane proteins. The automation of protein purification, and especially of the screening for optimal purification conditions will be addressed by the acquisition of an AKTA explorer chromatography system. This state-of-the-art system will carry out many tests of conditions without manual intervention, and will be used in a wide range of projects from directed evolution of new activities through the preparation of membrane proteins to aptamer purification. The recent acquisition in Leeds of a robot to perform X-ray crystallisation as been of tremendous benefit. However, we have found that a new bottleneck occurs during the optimisation of crystals after the initial conditions have been found. To remove this delay, we request here funds for the purchase of a robot to prepare the master blocks of solutions in which the additives, buffer and precipitant can be varied. This robot will find use in a wide range of crystallisation projects ongoing in Leeds, of which a few (involving directed evolution variants and membrane proteins) are specifically highlighted in this application. The projects described (from 7 academic staff) are all at a stage where the bottlenecks are limiting research progress. However, all other aspects of the described projects are in hand and with the funding of the requested instrumentation progress on these fundamentally important areas of research will be able to make rapid progress. In addition, the requested equipment will directly support the new Membrane Protein Structure Initiative (Mpsi), supported by the BBSRC SPoRT initiative, and involving 9 universities across the UK.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2004 (RE4) [2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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