PiMS at the OPPF Jon Diprose EMBO Course EBI, 23/09/2008

Overview ■The OPPF ■Workflows and Protocols ■Using PiMS at the OPPF

Aims of the OPPF ■To develop technology for high throughput protein production and crystallisation. ■To apply this technology to challenging biomedical problems through the development of a portfolio of collaborative projects. ■To make the technology available to the wider structural biology community.

Construct Design Cloning Purification Crystal Growth Crystallography… OPTIC Nautilus PlateDB Large-scale Expression Crystallogenesis The OPPF Pipeline Bioinformatics PCR Small-scale Expression Characterization

Construct Design Cloning Purification Crystal Growth Crystallography… OPTIC xtalP i MS Large-scale Expression Crystallogenesis The OPPF Pipeline Bioinformatics PCR Small-scale Expression Characterization

Overview ■The OPPF ■Workflows and Protocols ■Using PiMS at the OPPF

Workflows

Workflows in PiMS

PiMS Protocols

OPPF Protocols

Overview ■The OPPF ■Workflows and Protocols ■Using PiMS at the OPPF

Construct design ■Targets selected and constructs designed in Optic ■Imported into PiMS

PCR plate design ■ Plates designed in a spreadsheet template

Primer order plate ■

Primer order form ■

Operon order form page 1 ■

Operon order form page 2

Plate experiment workflow

Rescue plates ■Experiments that have not worked quite as expected can be repeated, maybe with a change to parameters and/or inputs.

Clean-up plates ■ Copy-from plate used to merge plates

Rescue and cleanup workflow

Scoring PCR plates

Scoring an expression plate

Purification protocols ■9 protocols to match Akta programmes ■Single column protocols ■Affinity with gradient elution ■Batch affinity ■Desalt ■Gel filtration ■Ion exchange ■Two column protocols ■Affinity desalt ■Affinity gel filtration ■Mammalian affinity gel filtration ■Mammalian affinity desalt

PiMS usage ■PiMS has been in use at the OPPF for 18 months ■Used exclusively to record wet-lab work this year ■221 Targets ■134 Plates ■544 Simple experiments

Acknowledgements › Dave Stuart (PI) › Ray Owens (Project Manager) › Robert Esnouf › Jon Diprose › Marc Savitsky (PiMS) › Ian Berry (now Evotec) › Joanne Nettleship › Louise Bird › Kim Henrick (PI) › Chris Morris (Project Manager) › Ed Daniels › Susy Griffiths › Bill Lin › Jo van Niekirk (now Ingres) › Anne Pajon › Peter Troshin