And now to something not so medical.... …but with the same biofilm techniques

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


Biofilm as the bad guy…


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


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


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


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.


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


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


Confocal laser scanning microscopy


Identification of variants- Sample from effluent tubes- Plating on selective media


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





Day 2 Day 3 Day 4 – 6 Day 7


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


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







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








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








Motility:

•Twitching (bottom) Swarming (top)

•Swimming (middle)

•Incubate ON at 30 °C








Crystal violet staining:•Wash away planktonic cells•Stain with 0.1 % CV•Wash wells•Dissolve stained biofilm with 96 % EtOH•Measure OD













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And now to something not so medical.... …but with the same biofilm techniques