Proteus slide show

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Biodeep October 12-15, 2003
Nature has more imagination than our dreams

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Contents

• Strains and clones transferred
• Microbial screening
• Proteases
• Esterases
• Xylanases
• Molecular screening
• Analysis of the cloned genomic fragments
• Construction/expression
• Biochemical characterization
• Biodeep Database
• Communications
• Conclusion

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Strains transferred

• 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
therefore stored in collection) and can be used
for future work
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Strains transferred

• Essex University (Dr. A. Sass)

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
therefore stored in collection) and can be used
for future work 3. The 19 strains of the 1st
batch which could not be cultured in our lab have
been sent a second time. The 40 strains of batch
3 are anaerobes (stricts or facultatives). 4
enrichment cultures currently used
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Strains transferred

• 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
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Strains transferred

• Messina University (Dr. G. Dauria)

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)
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Strains transferred

• 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
• about 70 pure strains growing in ONR medium will
  be transfer soon

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Strains transferred

• Review

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
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Clones transferred

• clones from TUB (Dr. P. Golyshin)
• 11 genomic fragments encoding 11 different
  esterases
• Corresponding (partial or FL) sequences have been
  provided with some genomic fragments
• Subcloning of oil8 have been done in fusion with
  pelB leader for periplasmic expression.
  Expression successful

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Microbial screening Protease

• Extracellular proteases using skim milk (0.2)
  screening assay
• Review (aerobes)

(1)
1. Planned with the pure strains to be transferred
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Microbial screening Protease

• Taxonomic position according Biodeep partners

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Microbial screening Esterase

• Esterases
• Principle of the CLIPS-O substrates

R C2H5 C9 H19
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Microbial screening Esterase

• Preliminary results Non induced (aerobes)
• Preliminary results Induced (aerobes).

1. Measured only using C10 CLIPS-O substrates
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Microbial screening Xylanase

• Xylanase using xylan blue (0.05) screening
  assay (xylan was used as carbon and energy
  sources)
• Preliminary results (aerobes)

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Microbial screening Xylanase

• Taxonomic position according Biodeep partners

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Molecular screening

• Proteases from strain P 1972 (Alteromonas sp.)
  and strain P 1600 (Bacillus 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 performed
• Positive hits identified and sequenced

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Analysis of the genomic fragment (PP 1972)
2989 bp
Identity/Similarity with Subtilisin (P00780) in
amino acids - FL gene 29-48 - Nterm
region of the gene 34-54
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Constructions
Construction Activity OK
OK
promoter
P1972 FL gene
1. 2.
pelB
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Subcellular localization
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Kinetic of expression
Induction at 28C
Induction
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Biochemical characterization
Thermostability at 30C
Thermostability at 40C
Thermostability at 30C
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Analysis of the genomic fragment (PP 1600)
4212 bp
Identity/Similarity with Intracellular Alkaline
Protease (P29140) in amino acids - FL gene
56-75
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Constructions
Construction Activity OK
In progress In progress
promoter
P1600 FL gene
Potential SP
pelB
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Biochemical characterization

• Optimum pH 8-9
• Other characteristics in progress

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Clones collection
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Biodeep database

• Protéus
• Design of the data sheet model
• Data related to 347 strains transferred in July
  to Univ. of Milano
• University of Milano
• Design of the database

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Communications

• Our industrial partners
• Congress
• MINATEC 2003 (Grenoble, France)
• BIO2003 (Washington, USA)
• INDUSTRIAL APPLICATIONS OF BIOCATALYSIS (Boston,
  USA)

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Conclusions and Outlook

• Current status of the Biodeep microbial
  collection 407 strains
• Current status of the Biodeep clones collection
  
• 13 cloned genomic fragments containing
  enzymatic activities (3 ORF subcloned)

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Timetable of future activities
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Thanks for your attention.
www.proteus.fr
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Biodeep database
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Biodeep database
These 2 forms have to be filled by each partner
for each strain