Basic Parts Update. Made 7 more parts since last week: cel9A, gliadin scFV, TevC, cel3A, OprF AtD, cl02365 AtD, Hia AtD Available passengers Gliadin scFv, Cub/Nub, Needle scFv, TevN/TevC, cellulases (cel3A, cel5B, cel9A, cel6A), SOD, Tir-M Available displayers
13 constructs from class: displayer, AG4/IILK, strep tag
96-well block – hard to see if all liquid aspirated out
Multichannel pipetting – inconsistent amount of liquid (and cells)
After 1 wash
Results consistent with previous experiment: A1 and A3 were brighter
Triplicates, all induced
– overnight incubation to saturation?
- the size and density of pellet seems to give a background so that smaller fluorescent pellets give similar readings as non-fluorescent larger pellets
Assays should measure the ability of Bacteria to degrade
Plate based assays exist:
-dyes form a colored complex with insoluble cellulose.
-Colonies that degrade insoluble cellulose remove color
from the plates.
-These methods are not very quantifiable.
Assays have been preformed with purified enzyme product:
-cellulose in filter paper is degraded into reducing sugar.
-dinitrosalicylic acid is added to the solution
-absorbance at 600 nm is measured
These assays are preformed at ph 4.8 in an acidic buffer
-will the cellulases be active under non acidic conditions?
-will our cells survive under acidic conditions?
gliadin scFv: To test whether scFv binds to gliadin
Mgfp-5: to test whether mgfp adheres to surfaces
- Place solution of cells (3ul) expressing mgfp-5 on glass slide
- incubate at room temperature for 10minutes
- Do successive washes with PBS to wash cells out.
- look at cells on glass slide under light microscope
- measure cell density
- positive control: glass-adherent bacteria(E. coli B117)
- negative control: solution of cells not expressing mgfp-5
- PMID: 16979252