Drummond:Paromomycin
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Introduction
Error spectrum
- binds to A-site of ribosome
- in yeast, paromomycin acts by blocking aminoacyl-tRNA binding to A-site of ribosome after initiation
- interrupts with elongation step of translation [1]
Dose response
Stock solution
- Paromomycin is water soluble (50 mg/mL) (Sigma Aldrich)
- in the lab, a 100 mg/mL stock solution of paromomycin was synthesized (some vortexing was necessary)
Paromomycin dilution series
- 100X paromomycin stock solution (for 140 μM)
- 140 μM paromomycin for 50% cell growth inhibition [1]
- (1.4*10^-4 mol paromomycin/L solution)(713.71 g/mol)(1000 mg/g)(1 L/1000 mL) = 0.0991 mg/mL ≈ 0.1 mg/mL
- (0.1 mg paromomycin/mL sln.)(50 cultures)(2 mL sln./culture) = 10 mg paromomycin (for 100 mL solution [2 mL*50])
- 100 mL/100 = 1 mL
- 10 mg paromomycin/1 mL nanopure water for 100X stock solution
- 2-fold dilution series (1:1 to 1:64)
- 500 μL 100X paromomycin stock solution into tube 1 (1:1 dilution)
- 250 μL nanopure water into 6 tubes (1:2 to 1:64)
- Pipet 250 μL from 1:1 dilution into 1:2 dilution
- Pipet 250 μL from 1:2 dilution into 1:4 dilution
- " 1:4 to 1:16, 1:16 to 1:32, 1:32 to 1:64
For the second paromomycin trial, we adjusted the range of concentrations of paromomycin in which the yeast cells were grown. Instead of a 100X stock solution of paromomycin, a 1000X stock was used.
- 1000X paromomycin stock solution (for 140 μM)
- 62.0 mg paromomycin/620 μL H2O
- vortex solution to dissolve paromomycin
- 1:1 to 1:2048 serial dilutions
- 100X Yeast preculture stock solution
- Maximum OD after 21 hours (14 doublings) = 10
- 10/214 = 6.10*10-4
- 6.10*10-4/Initial preculture OD = X mL preculture (in 1 mL YPD solution)
- Yeast growth with paromomycin
- 1.8 mL YPD
- 200 μL paromomycin dilution (1:1 to 1:2048) or H2O for controls
- 20 μL 100X yeast culture
- incubate overnight at 30°C
- Record OD as a percentage of the control (0 μM paromomycin)for each dilution
cooperativity: 1.329
half max: 305.968
(using R and hill equation)