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CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare

Daniele Daffonchio Tullio Brusa Sara Borin. Adriana Bona Enrica Canzi Claudia Sorlini. WP6 : genetic characterization of isolated microorganisms and of enzymes interesting for biotechnological application. Partner 1c. CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare.

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CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare

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  1. Daniele Daffonchio Tullio Brusa Sara Borin Adriana Bona Enrica Canzi Claudia Sorlini WP6: genetic characterization of isolated microorganisms and of enzymes interesting for biotechnological application Partner 1c CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare DISTAM Università degli Studi di Milano

  2. Isolated strains Research for xyl E-type genes (catechol 2,3 dioxigenase) gene via PCR All negatives Community DNA • DHABs cultivable/uncultivable bacteria do not clevage aromatic ring via common extradiol cleavage Catabolic genes characterization 19% of the isolates are able to grow on oil and catechol as unique carbon sources

  3. Plasmid profile characterization Plasmid profile has been characterised for several 2001 aerobic isolates • Plasmids (mobile genetic elements?) are abundant among Discovery isolates, and absent in L’Atalante strains • Plasmids are equally distributed between the most represented phylogenetic groups

  4. donor MATING host Screening for: Hg resistance NaCl tolerance Oil degradation Naphtalene degradation Search fortransconiugants NEGATIVE “Fishing” of functional genes in the metagenomic DNA Plasmids donor: cells recovered filtrating Discovery water/brines interface concentrated Plasmids exogenous isolation Plasmids hosts: Pseudomonas putida

  5. Screening for natural competent strains • Horizontal gene transfer (HGT) occurs in many natural environments • Natural competent cells are naturally able to acquire naked DNA via natural transformation • Extreme conditions imposed by the brines could stimulate competent cells to exchange DNA in order to adapt to the environment In DHABs, is there free DNA? In DHABs, are there natural competent bacteria? Could brines stimulate the competence in these strains? Could brines enhance natural transformation and HGT?

  6. 2ng 250ng 1min 8days 50ng 6hours 10ng 2days 1day DNA stability in brines • DNA is detectable in Discovery brine for at least 1 day: after exposure to brines DNA maintains the trasforming ability?

  7. Plating on Ap and Km Screening for ApR KmR colonies Incubation with pZR80 Washed cells ApS, KmS Natural competent strain • Trasformation experiments • donor plasmid pZR80: • broad host range plasmid • confers resistance to ampicillin and kanamycine host strains: • isolated strains of the -proteobacteria group

  8. Natural competent strain? No plasmid could be detected in Ap/Km resistant strains Trasformation experiments 5D 18B Alteromonas macleodii Halomonas meridiana NaCl 5% 0.E+00 3.E-08 PCB 0.E+00 8.E-10 OTB 0.E+00 1.E-08 filter 1.E-09 1.E-09 brine 0.E+00 8.E-05

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