DNA Microarray Technology & Bio-Informatics. Forbedring af produktions mikroorganismer

Transkript

1 DNA Microarray Technology & Bio-Informatics Forbedring af produktions mikroorganismer

2 DNA Microarrays forstå og forbedre produktions stammer: på cellulært niveau se hvad der sker I cellen Wild-type cells Mutants Pathogenic strains Developmental processes Culture conditions Gene function Gene discovery Gene regulation Signal transduction Pathway elucidation Differentiation cascades

3 DNA Microarray fremstilling anbringelse af et genom påp 1 Chip microskop glas PCR produkter oligoes coated glas poly-lysine Arrayer Printing (Arraying) B. subtilis Chip

4 Sammenlignelig Hybridisation cultures A B Scanner Total RNA cdna,, label Cy3 Cy5 hybridisation, washing Image analysis: : Scanning

5 DNA Microarray Technology What genes are turned off at the end of exponential growth? Gene expression high enzyme production low enzyme production Hybridized to Cy3 probe from log cells (control) Cy5 probe from stationary phase cells

6 DNA micro array application to the HA Project Red pigment is a potential problem for recovery First seen at 12 hours in a fermentor, the colour intensifies as fermentation progresses Not produced by lower yielding strain RB161 Not separated by centrifugation Some pigment stays in supernatant with HA

7 Bevis for at microarray information er brugbar!! Wild-type HA fermentation yvmabc/cypx strain Novozymes fokus er bla. at identificere gener der forøger udbyttet. Vi har micorarrays for vores vigtigste produktions værter.

8 Nano-teknologi initiativer til måling på molekylært niveau: - mechanistic input to protein engineering, assay and application optimization Coordinated by Thomas Callisen,, Allan Svendsen.

9 Study of structure-function relationships of enzymes at interfaces Dynamics of surface exchange processes: surface plasmon resonance (SPR) pg/mm2 resolution via in-house automated micro-fluidic chip technology Surface topology: atomic force microscopy (AFM) nm resolution Single molecule diffusion of at interface and in bulk: fluorescence confocal microscopy (CLSM) Interactions in bulk: dynamic lightscattering (DLS), isothermal calorimetry (ITC), cryo-transmission electron microscopy (cryo-tem)

10 Example 1: Protease activity Max height (nm) Spin coated film of blood (a 2 um x 2 um picture, 5 nm deep hole has been scraped by the AFM tip) Time (min) Washed with buffer Real-time monitoring by AFM of proteolysis of hemoglobin (Imaging in tapping mode before picture analysis). - collaboration w. Nano Science Center at KU (1 Response unit ~ g/mm2 ) RU association of substrate (albumin) Substrate pulse Time net effect of association/activity of 4 protease variants Enzyme pulse SPR detection of adsorption of protein substrate followed by protease association and degradation as function of the enzyme variant Change in adsorbed mass s

11 Example 2: Cellulase activity RU 400 Celluzyme (2-16 mg/l) injected over spin-coated cellulose surfac 0.35 Celluzyme activity (ug/m2/s) towards spin-coated cellulose su Response Time s Cellulase binding and activity towards a 60 nm cellulose film prepared by spin-coating - collaboration w. Surface Chemistry Institute (YKI)

12 Yellow craters appear in the lipid surface, where the lipase has degraded the lipid Insert: Activity of lipase pr. time measured using the crater area -2.miṉ1 ) Hydrolysis rate of HLL on monomoleculat films of 1-MO or [DMPC/DMPG(70:30)]/[1-MO](70:30) 1-MO [DMPC/DMPG(70:30)]/[1-MO](70:30) Buffer: 10 mm Tris/HCl (ph 8.0 [HLL] = 0.16 nm [ß-CD] = 0.8 mg.ml surface pressure (mn.m -1 ) Balashev, K.; Nielsen, L. K.; Callisen, T. H.; Svendsen, A.; Bjørnholm, T. In Lipases and Lipids: Structure, function and Biotechnological Applications; Kokotos, G., Constantinou-Kokotou, V., Eds.; Crete University Press: 2000; pp

13 Nano wash analysis. Atomic Force Microscope images 0 min 5 min 15 min 30 min 50 min 75 min 100 min 130 min Fig.3 AFM images of a time sequence of the HLL hydrolysis of supported mixed DPPC:DPG (70:30 (%)) bilayer on mica obtained from collapsed vesicles. (A) images obtained after injection of 145 nm HLL solution.

14 Nano Wash Kinetics t = 0 t = 17 t = 22 t = 24 t = 25 t = 27 DPPC/ 35 mn/m / PLA2 / ph = 8 (HEPES)/ F < 1 nn / 5 Hz / 25 C / 5 x 5 µm lag phase despite defects product domaine formation during lag phase burst initiated at product domains Kildemark/Risbo

15 HLL in interaction with the DLG lipid.

16 Cartoon of the orientation of HLL on POPG vesicles as deduced by the ESR quenching experiments.

17 Forståelse af enzym funktion og substrat interaktion på molekylært niveau: Giver en unik mulighed for at udvikle bedre assays og hermed bedre enzymer. Kan blive starten til en revolution indenfor enzymatisk katalyse: koblede enzym funktioner (føder hinanden, minimere diffusion => mange gange hurtigere) koblet funktionalitet: performance materialer og enzym(er)

18 Forståelse for protein / protein eller andre molekylære interaktioner samt molekyleres aggregering ( self assembling): vigtigt for udvikling af biosensorer vigtigt for biomateriale udvikling vigtigt for bio chip/ computer chip udvikling

19 Fremtid: Gæt på udvikling indenfor White Biotechnology bla baseret på udvikling af nano teknologiske metoder: McKinsey rapport fra 2003/4 forudsiger en kraftig vækst!

20 White biotechnology now ready for take-off Market penetration Examples of white biotechnology Bio-based polymers Fuel from agriculture waste Biological processes for vitamin production Today White biotechnology is the application of nature's toolset to industrial production Time Industrial enzymes e.g.: detergent,textile, pulp&paper,, waste, food, feed

21 10-20% impact on chemical industry in 2010 Source: McKinsey McKinsey analysis: Chemical industry impacted by biotechnology 20% 10% 2000 Today 2010 Fast uptake Slow uptake Time Impact* ) dependent on: Technology development Overall demand Feedstock prices Policy framework * ) Impact means the use of biotechnological process steps such as fermentation, biocatalysis, etc.

22 Source: McKinsey Economy: EUR billion annual added value by 2010 Impact of white biotechnology Cost reduction Raw materials Process costs Investments McKinsey estimate of annual added value by the global chemical industry EUR billions 6-12 Additional revenues New products 5-10 Value-added added processes

23 Environment: Decreasing the footprint Impact of white biotechnology Increased energy and process efficiency Renewable feed- stock Reduction of: Greenhouse gas emissions Emissions to water Emissions to air Resource usage

24 Konklusion : hvad driver nanotek.. udvikling? hvad er realistisk og hvad er science fiktion? ufattelig svært at spå om fremtidig værdi! Nano teknologi vil medføre udvikling af essentielle redskaber til at accelerere udvikling af bedre produkter indenfor bioteknologi. Nye metoder vil muliggøre den nødvendige effektivisering, så nye store markeder åbnes for biokatalyse (fuel alcohol, waste treatment, polymer syntese, fine chemical ). Vanskelig at spå om bio chips/ biomaterialers realistiske muligheder det går nok langsommere end man vil forvente pga pris. Genteknologi bliver nok forsat den mest værdifulde nanoteknologi indenfor Bioteknologi (og udviklingen har taget mange år). Den største enkelt værdi af ny nanoteknologi kan sandsynligvis skabes indenfor farma formulering og diagnostik udvikling.