Biodeep

May 12-14, 2003

“Nature has more imagination

than our dreams”

Contents

Strains and clones transferred Microbial screening

Proteases Esterases

Molecular screening Biodeep Database Communications Conclusion

Strains transferred

Milan University (Dr. T. Brusa)

Culturesreceived

Strainsstudied(1)

Strainscultures(2)

1st Batch 94 94 892nd Batch(anaerobes) 18 18 18

Total 112 112 107

1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work

Strains transferred

Essex University (Dr. A. Sass)Culturesreceived

Strainsstudied(1)

Strainscultures(2)

1st Batch 117 117 98(3)

2nd Batch 90 63 63

Total 207 180 161

Anaerobic strains will be transfer soon1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work3. The 19 strains of the 1st batch which could not be cultured in our lab have been sent a second time recently

Strains transferred

Messina University (Dr. G. D’auria)Culturesreceived

Strainsstudied(1)

Strainscultures(2)

1st Batch 59(3) 37 18

2nd Batch 42(3) 34(4) 4

Total 101(3) 71 22

1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work3. See next slide4. All the strains excepted the ones growing in P10 medium (petrol based medium)

Strains transferred to Protéus Cultures from Univ. Messina

Enrichment cultures and pure strains No synthetic media defined (from the water point of view) We tried to grow the stains in a defined medium The growth conditions defined for the 22 strains cultured in our lab are available

Strains transferred

LLM (Dr. F. Morel)

Culturesreceived

Strainsstudied(1)

Strainscultures(2)

1st Batch 20 20 20

Total 20 20 20

1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work

Strains transferred

Biodeep Partners Culturesreceived

Strainsstudied (1)

Strainscultured (2)

Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)

11220710120

1121807120

1071612220

Total 440 383 310

1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab and can be used for future work

Review

Clones transferred clones from TUB (Dr. P. Golyshin)

3 genomic fragments encoding 3 different esterases (oil2, oil7 and oil8) Corresponding sequences have been provided with the genomic fragments subcloning of oil8 in progress for shuffling purposes

Microbial screening Extracellular proteases using skim milk (0.2%)

screening assay Examples

P 1600 P 1975 P 1972

After 3 days at 25°C After 2 days at 25°C

Biodeep Partners Culturesscreened

Positivecultures

Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)

1261012

102704

Total 85 41

Review (aerobes)

Results

Results

Taxonomic position Culturesscreened

positivecultures

Actinobacterium sp.Alpha proteobacteria sp.Alteromonas sp (inclusing P 1972)Bacillus sp. (inclusing P 1600)Halobacillus sp.Halomonas sp.Idiomarina sp.Marinobacter sp.Pseudomonas sp.Thiobacillus sp.Salmonella sp.Vibrio sp. inclusing P 1975)Virgibacillus sp.

228174411441121

10713101010021

Unidentified strains 24 14Total 85 41

Taxonomic position according to the studies done

by each Biodeep partners

Microbial screening Esterases Principle of the

CLIPS-O substratesR = CH3 C2H5 C9H19 C15H31

(1)(2)(3)(4)

O

OH

O

NO2

R

O

O

OH

HO

NO2

BSA

O

ONO2

H

H

O

H

HO

NO2

beta-elimination(BSA)

chemical oxidation(NaIO4)

Measurable at 414 nm

esterase / lipase

R = C2H5

C9 H19

Preliminary Results

Biodeep Partners Culturesscreened

Positivecultures

Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)

0600

0200

C3>C10

Total 6 2

Review (aerobes)

Molecular screening

Proteases from strain P 1972 (Alteromonas sp.)

Genomic library construction : partial restriction

using Sau 3A I

Inserts size ranging from 2 to 5 kb

Quality control : less than 10% of empty clones

Screening results

Library screening

12 000 clones screened

6 (confirmed) positive hits

Expression (preliminary results)

Negativecontrol

Clone 9Clone10

Biodeep database

Protéus

Design of the data sheet model Model sent to University of Milano on February, 2002

University of Milano

Design of the database

Biodeep databaseGeneralities

Strain Name :Original lab :Scientific corresponding :Origine of the sample : basins and brine or sea water or interfaceReference culture medium See next pagePreservation conditions Cryoprotector :

Taxonomic position : Genus and specie Identification method : seq 16S, hybridization DNA/DNA…Publication : Yes/No References :

Physiological and metabolical caracterization of the strain

Microscopic observations :Mobility :Gram :Sporulation : Analytical methodGC% content: Analytical method

Growth conditions Range OptimumTemperatureSalinity (g/l)pH

Oxydative metabolism : Aerobic strain, Anaerobic strains (electron acceptors), Microaerophilic strain

Substrates used :Substrates not used :Enzymatic activitiesMetabolites :Sequences done and function of this sequences :

Remarks :

Biodeep databaseMedium Name :Reference (if possible)

Composition for 1L :Components Units (g, mg ou mL)________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______________________ _______

Adjust pH to : With :

Sterilisation procedure

Solutions to prepare and to add separatly

Remarks :

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

Biodeep database

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

Data to be transferred to Protéus

Protéus will transfer all the data to University of Milano

Integration of all the data by University of Milano

Biodeep database

Up to now, we received from University of Marseille 20 forms filled.

Communications

Our industrial partners

Congress : EMBO workshop (France) INFORMEX (USA) BioMarket 2002 (England) BIOTECNICA Business Forum (Germany) Global Venture Forum (Japan)

Conclusions and Outlook Current status of the Biodeep collection :

310 strains

Identification of strains producing esterases

and extracellular proteases

Protease gene(s) from strain P 1972

(Alteromonas sp.) already cloned (sequencing in

progress)

Database ready to be filled?

New strains and bulk DNA?

Timetable of future activities

Activities AvailableMicrobial data base for partners Before the end of year 2002

Culture collection (1st cruise) End of year 2002First screening for products During year 2003

Culture collection (2nd cruise) End of year 2003

…Thanks for your attention.

www.proteus.fr

Environmental libraries

Samples Status

Lib 1 Atalante Interfacesample

Completed andvalidated

Lib 2 Urania Interfacesample

Completed andvalidated

Lib 3 Enrichment culturefrom Urania

Interface sampleIn progress

Lib 4 Enrichment culturefrom Urania

Interface sampleIn progress