Drummond:Paromomycin

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 Tselika-Biochimie-2008

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)
 * 1) 140 μM paromomycin for 50% cell growth inhibition Tselika-Biochimie-2008
 * 2) (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
 * 3) (0.1 mg paromomycin/mL sln.)(50 cultures)(2 mL sln./culture) = 10 mg paromomycin (for 100 mL solution [2 mL*50])
 * 4) 100 mL/100 = 1 mL
 * 5) 10 mg paromomycin/1 mL nanopure water for 100X stock solution
 * 2-fold dilution series (1:1 to 1:64)
 * 1) 500 μL 100X paromomycin stock solution into tube 1 (1:1 dilution)
 * 2) 250 μL nanopure water into 6 tubes (1:2 to 1:64)
 * 3) Pipet 250 μL from 1:1 dilution into 1:2 dilution
 * 4) 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)
 * 1) 62.0 mg paromomycin/620 μL H2O
 * 2) vortex solution to dissolve paromomycin
 * 1:1 to 1:2048 serial dilutions


 * 100X Yeast preculture stock solution
 * 1) Maximum OD after 21 hours (14 doublings) = 10
 * 2) 10/214 = 6.10*10-4
 * 3) 6.10*10-4/Initial preculture OD = X mL preculture (in 1 mL YPD solution)


 * Yeast growth with paromomycin
 * 1) 1.8 mL YPD
 * 2) 200 μL paromomycin dilution (1:1 to 1:2048) or H2O for controls
 * 3) 20 μL 100X yeast culture
 * 4) incubate overnight at 30°C
 * 5) 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)