Strep Mutations Data Sheet

Strep Mutations Data Sheet - Genetics - BIOL302L...

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Genetics - BIOL302L Streptomycin mutations: Isolation, Characterization and Analysis Progress Report Name: ___ Section: _ Objective The purpose of this experiment was to determine the frequency of spontaneous mutations occurring in the rpsL gene coding for rpS12, a ribosomal protein to which streptomycin binds. In order to obtain spontaneous strep mutants of e. coli, we observed growth of colonies on TBAB and TBAB + Strep plates, and the mutants from the TBAB + Strep plates were then transferred to a new TBAB + plate and a TBAB plate in order to determine whether the mutants were strep resistance, intermediate, or dependence. E.coli is Strep R if streptomycin cannot bind to the rpS12, and is Strep D if the cell dies without the streptomycin being bound to rpS12. By using the polymerase chain reaction, the rpsL gene was amplified and then sequenced. The sequencing process helped determine the type and the location of the mutation in our colonies. In the sequencing process, we used dideoxy-nucleotides, which terminate extension and have a different colored fluorescent tag for each, which indicates exactly where the mutation occurred. We were then able to compare our sequence to that of the wildtype and those of our classmates. Data: TBAB + Strep __3 ___ No Strep, 10 - 6 Dilution __107 __ __ Avg__114.5 __ How many of the clones on the strep plate were: Resistant: __1 __ Dependent: __0 __ Intermediate: __2 __ Calculations: Viable Cell Count: (Avg. # of two TBAB/0.1mL) x 10 6 = (114.5/0.1 mL) x 10 6 = 1.1145 x 10 9 cfu/mL Str R , Str D , and Str I Mutant Concentrations: (# of Str R / original volume tested) = (1 colony / 1.5 mL) = 6.7 x 10 -1 mutants/mL (# of Str D / original volume tested) = (<1 colonies / 1.5 mL) =
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< 6.7 x 10 -1 mutants/mL (# of Str I / original volume tested) = (2 colonies / 1.5 mL) = 1.33 mutants/mL Str R , Str D , and Str I Spontaneous Mutation Frequencies: Str R : (Mutant Concentration / Viable Cell Count) = (0.67 mutants/mL / 1.1145 x 10 9 cfu/ mL) = 6.012 x 10 -10 mutants/cfu Str D : (Mutant Concentration / VCC) = (<1 mutants/mL / 1.1145 x 10 9 cfu/mL) = <6.012 x 10 -10 mutants/cfu Str I : (Mutant Concentration/ VCC) = (1.33 mutants/mL / 1.1145 x 10 9 cfu/mL) = 1.193 x 10 -9 mutants/cfu
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Strep Mutations Data Sheet - Genetics - BIOL302L...

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