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And now to something not so medical.... …but with the same biofilm techniques. Diversity in Pseudomonas putida biofilm. Biofilm as an industrial capacity Exercise 4 Anne-Mette Juel Christensen PhD Student, Infection Microbiology group August 2014. Biofilm as the bad guy…. - PowerPoint PPT Presentation
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17/04/2008Presentation name1 DTU Systems Biology, Technical University of Denmark
And now to something not so medical....
…but with the same biofilm techniques
Diversity in Pseudomonas putida biofilm
Biofilm as an industrial capacity
Exercise 4
Anne-Mette Juel ChristensenPhD Student, Infection Microbiology group
August 2014
17/04/2008Presentation name3 DTU Systems Biology, Technical University of Denmark
Biofilm as the bad guy…
17/04/2008Presentation name4 DTU Systems Biology, Technical University of Denmark
Biofilm as the bad guy…• Surfaces – a space to be occupied!• Localizing cells in close proximity
– Communication– Exhange of genetic material -> establish niche growth
• Protection from environmental challenges e.g. antibiotics, host immune response, toxic compounds
• Extracellular polymeric substances (EPS) forms a barrier• Creation of dorment cells• Differentiation of cells into subpopulations e.g. non-motile ->
heterogeneity
-> Robust community of cells that causes problems for the host
17/04/2008Presentation name5 DTU Systems Biology, Technical University of Denmark
Biofilm as the good guy…-> Robust community of cells that can be…
– Resilient to variety of environmental stress – Potent biocatalyst of toxic and insoluble products
In industry:•Microbial subpopulations in industrial fermentations
– Undesirable bi-products– Alter substrate uptake– Growth reduction– Alter metabolic activities
17/04/2008Presentation name6 DTU Systems Biology, Technical University of Denmark
Exercise 4: Diversity in P. putida biofilm• Industrial relevant production organism – Pseudomonas putida– Non-pathogenic– Biofilm capability– Produce compounds interesting for biofuel production ->
isobutanol– Isobutanol toxic to the cell
• Pseudomonas sp. VLB120 - originally isolated from forest soil- has shown to be able to grow on a wide range of carbon sources- grow in the presence of different solvents e.g. toluene and
styrene - GFP tagged
17/04/2008Presentation name7 DTU Systems Biology, Technical University of Denmark
Aim of the exercise
•Study diversification and change of biofilm structure of Pseudomonas sp. VLB120 biofilm when exposed to 2.5 % isobutanol during growth in FB medium with citrate as carbon source in the flow chamber system.
17/04/2008Presentation name8 DTU Systems Biology, Technical University of Denmark
Experimental procedure
Assembly of the biofilm system, preparation of medium +/- isobutanol
Sterilization and washing. Preparation of cultures
Inoculation of flow chamber channels
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Done for you
17/04/2008Presentation name9 DTU Systems Biology, Technical University of Denmark
Biofilm set up
FB medium + 1 mM citrate
FB medium + 1 mM citrate
+ 2.5 % isobutanol
Each team has one flow cell with.
-2 channels treated with FB medium + citrate + 2.5 % isobutanol and 1 channel treated with FB medium + citrate
OR
- 1 channel treated with FB medium + citrate + 2.5 % isobutanol and 2 channels treated with FB medium + citrate
17/04/2008Presentation name10 DTU Systems Biology, Technical University of Denmark
Confocal laser scanning microscopy
17/04/2008Presentation name11 DTU Systems Biology, Technical University of Denmark
Identification of variants- Sample from effluent tubes- Plating on selective media
17/04/2008Presentation name12 DTU Systems Biology, Technical University of Denmark
Day 2 – Monday 11th august
•Morning: – CLSM– Prepare LB PIA plates
•Afternoon:– CLSM cont.– Collect effluent in epp. tubes – keep sterile!– Make dilutions in 0.9 % NaCl and plate on LB PIA plates– Incubate ON at 30 °C
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7
17/04/2008Presentation name13 DTU Systems Biology, Technical University of Denmark
Slight change of experimental set-up• Flow cell 1
– VLB120 wt– VLB120 wt– VLB120 + isobutanol from t=0
• Flow cell 2– VLB120 wt– VLB120 + isobutanol from t=0– VLB120 + isobutanol from t=0
• Flow cell 3– VLB120 wt– VLB120 wt– VLB120 + isobutanol from t=0
• Flow cell 4– VLB120 wt + isobutanol from day 4– VLB120 + isobutanol from t=0– VLB120 + isobutanol from t=0
• Flow cell 5– VLB120 wt + isobutanol from day 4– VLB120 wt + isobutanol from day 4– VLB120 + isobutanol from t=0
Only microscopy and effluent collection on VLB120 wt and VLB120 wt + isobutanol from day 4
17/04/2008Presentation name14 DTU Systems Biology, Technical University of Denmark
Day 4 (day 3 in manual)• Team 1-3 -> Pseudomonas sp. VLB120 wt• Team 4-5 -> Pseudomonas sp. VLB120 treated with 2.5 % isobutanol at
day 4
• Look for variants on plates• Take pictures (1.st floor, lab 101 – Leica)• Select 4 variants and re-streak on LB PIA plates• Incubate ON at 30 °C• Keep original plates at room temperature
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7
17/04/2008Presentation name15 DTU Systems Biology, Technical University of Denmark
Day 5 (day 4 in the manual)
Stability:•Compare original plates (pictures) with re-streak. Stable?•Leave plates at room temperature (to be used for motility)
Growth study and crystal violet biofilm staining (CV staining)•If stable inoculate single colony in 10 mL LB• Incubate ON at 30 °C• Prepare FB medium + 1mM NaCitrate
Motility (Swimming, swarming & twitching):•Prepare 5 plates for each motility type
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7
17/04/2008Presentation name16 DTU Systems Biology, Technical University of Denmark
Day 6 (day 5 in the manual)
Growth study:•50 mL LB medium in 100 mL flasks•Measure OD600 on ON culture -> inoculate to a conc. Of OD600=0.01•Measure OD600 at appropriate intervals e.g. every hour•Calculate doubling time and compare with wt
Crystal violet staining:•Dispense 140 uL in all wells.•Add 10 uL ON cultures•Remember blanks
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7
17/04/2008Presentation name17 DTU Systems Biology, Technical University of Denmark
Day 6 (day 5 in the manual)
Motility:
•Twitching (bottom) Swarming (top)
•Swimming (middle)
•Incubate ON at 30 °C
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7
17/04/2008Presentation name18 DTU Systems Biology, Technical University of Denmark
Day 7 (day 6 in the manual)
Crystal violet staining:•Wash away planktonic cells•Stain with 0.1 % CV•Wash wells•Dissolve stained biofilm with 96 % EtOH•Measure OD
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7
17/04/2008Presentation name19 DTU Systems Biology, Technical University of Denmark
Day 7 (day 6 in the manual)
CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells
Select morphology variants for phenotypic characterization
Phenotypic characterization: Stability, growth, CV staining of biofilm, motility
COMSTAT and Imaris analysis
Day 2 Day 3 Day 4 – 6 Day 7