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The growth characteristics of each mutant will be tested using plating
assays. Each mutant will be tested on two different media (YPD, rich
media and YNB, minimal media) at 3 different temperatures (24, 30 and
24 degrees C). If the knockouts are unable to grow on minimal media,
they will be tested with a battery of plating assays to determine which
auxotrophy they possess.
Cell-wall inhibition
Mutants will be screened for defects in cell-wall
integrity using a variety of cell-wall inhibitors and cell-wall biogenesis
inhibitors. We have begun plating assays using the following cell-wall
inhibitors:
-Flourescent Brightner 28 - Sigma F-3397/(calcofluor
white)
stock soln: 15 mg/ml in H2O
concentrations: 0.5, 1.0 & 1.5 mg/ml
function: binds to chitin
-SDS (sodium
dodecyl sulphate; AnalaR Biochemical; BDH Chemicals Ltd., Poole, England)
stock soln: 10% in H2O
concs.: 0.01%, 0.03% & 0.06%
function: cells with compromised cell wall integrity more sensitive
-Congo red -
Sigma C-6767
stock soln: 4% in 50% ethanol
concs.: 0.5%
function: binds to beta-(1,4) glucans & interferes with cell wall
constuction
-Caffeine -
Sigma C-0750
stock soln: 10 mg/ml in H2O
concentrations: 0.2, 0.5 & 1.0 mg/ml
function: inhibits signal transduction pathways important for cell-wall
biogenesis
Other in vitro studies to be done:
-Cell wall biogenesis inhibitors
-Vanadate resistance
-Osmoloarity studies (survival in H2O vs. sorbitol)
-Trypan blue uptake (cells with compromised cell wall integrity should
internalize dye faster/at a higher rate than wt)
-Antifungal susceptibility testing
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