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

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

3. 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. http://www.oppf.ox.ac.uk/

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

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

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

7. Workflows

8. Workflows in PiMS

9. PiMS Protocols

10. OPPF Protocols

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

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

13. PCR plate design ■ Plates designed in a spreadsheet template

14. Primer order plate ■

15. Primer order form ■

16. Operon order form page 1 ■

17. Operon order form page 2

18. Plate experiment workflow

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

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

21. Rescue and cleanup workflow

22. Scoring PCR plates

24. Scoring an expression plate

26. 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

27. 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

28. 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