1. Biodeep May 12-14, 2003 “Nature has more imagination than our dreams”

2. Contents l Strains and clones transferred l Microbial screening 3 Proteases 3 Esterases l Molecular screening l Biodeep Database l Communications l Conclusion

3. Strains transferred l Milan University (Dr. T. Brusa) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work

4. Strains transferred l Essex University (Dr. A. Sass) Anaerobic strains will be transfer soon 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work 3. The 19 strains of the 1 st batch which could not be cultured in our lab have been sent a second time recently

5. Strains transferred l Messina University (Dr. G. D’auria) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work 3. See next slide 4. All the strains excepted the ones growing in P10 medium (petrol based medium)

6. Strains transferred to Protéus l Cultures from Univ. Messina 3 Enrichment cultures and pure strains 3 No synthetic media defined (from the water point of view) 3 We tried to grow the stains in a defined medium 3 The growth conditions defined for the 22 strains cultured in our lab are available

7. Strains transferred l LLM (Dr. F. Morel) 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work

8. Strains transferred 1. Strains studied means that we tried at least once to culture them 2. Strains cultured means strains that have been cultured in our lab and can be used for future work l Review

9. Clones transferred l clones from TUB (Dr. P. Golyshin) 3 3 genomic fragments encoding 3 different esterases (oil2, oil7 and oil8) 3 Corresponding sequences have been provided with the genomic fragments 3 subcloning of oil8 in progress for shuffling purposes

10. Microbial screening l Extracellular proteases using skim milk (0.2%) screening assay l Examples P 1600 P 1975 P 1972 After 3 days at 25°C After 2 days at 25°C

11. l Review (aerobes) Results

12. l Taxonomic position according to the studies done by each Biodeep partners

13. Microbial screening l Esterases l Principle of the CLIPS-O substrates R = C 2 H 5 C 9 H 19

14. Preliminary Results l Review (aerobes)

15. Molecular screening l Proteases from strain P 1972 (Alteromonas sp.) l Genomic library construction : partial restriction using Sau 3A I l Inserts size ranging from 2 to 5 kb l Quality control : less than 10% of empty clones

16. Screening results l Library screening 312 000 clones screened 36 (confirmed) positive hits l Expression (preliminary results) Negative control Clone 9 Clone 10

17. Biodeep database l Protéus Design of the data sheet model Model sent to University of Milano on February, 2002 l University of Milano Design of the database

18. Biodeep database

19. These 2 forms have to be filled by each partner for each strain

20. Biodeep database l Data form to be completed by each partner For the isolated strains And update of the form as soon as new information and results are obtained l Data to be transferred to Protéus l Protéus will transfer all the data to University of Milano l Integration of all the data by University of Milano

21. Biodeep database l Up to now, we received from University of Marseille 20 forms filled.

22. Communications l Our industrial partners l Congress : 3 EMBO workshop (France) 3 INFORMEX (USA) 3 BioMarket 2002 (England) 3 BIOTECNICA Business Forum (Germany) 3 Global Venture Forum (Japan)

23. Conclusions and Outlook l Current status of the Biodeep collection : 310 strains l Identification of strains producing esterases and extracellular proteases l Protease gene(s) from strain P 1972 (Alteromonas sp.) already cloned (sequencing in progress) l Database ready to be filled? l New strains and bulk DNA?

24. Timetable of future activities

25. …Thanks for your attention. www.proteus.fr

26. Environmental libraries