Post on 12-Jan-2016
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