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This page was written to fulfill a requirement for Genetics Lab at UMBC
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Cristina Casaje: Sec. 302, Writer, Editor, Liason
Venice Luceriaga: Sec. 203, Writer, Web Developer
Created December 14, 2004
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Experiment: Streptomycin Mutations in
E. coli
Discussion
To identify spontaneous mutations, E. coli was spread on two different plates: TBAB and TBAB+Strep. The TBAB plates produced an average of 118.5 colonies. The TBAB+Strep plates produced 4.5 colonies. These colonies are the mutant colonies. The mutation frequency was calculated to be 9.1x10-9 cfu/ml. This mutation is very small saying that in 1.19x109 cfu/ml viable cells there is a probability of 9.1x10-9 that a mutation would occur. Mutation frequency was low because of proofreading mechanisms that corrects errors in transcription. This mutation has been concluded to be a missense mutation- in which a codon is substituted and a new amino acid is coded for. This was determined by ruling out the other types of point mutations. The presence of the resistant colonies rules out the possibility of neutral and silent mutations because the protein has been altered to prevent binding of Streptomycin. A nonsense mutation or frameshift would have resulted in nonsense codons and the gene would have been ‘knocked out’ and no growth would have appeared.
With the mutation type identified there was still the question of our mutation being streptomycin dependent or resistant. A colony from the TBAB+Strep plate was scored onto a new TBAB plate and TBAB+Strep plate and was incubated overnight. E. coli growth was found on both plates. This proved that our E. coli sample was Streptomycin resistant. If there was no growth on the TBAB plate this would’ve meant that the presence of Streptomycin was needed for growth- ‘Streptomycin dependent.’ Since mutants grew on both plates we concluded that Streptomycin is insignificant- ‘Streptomycin resistant.’
For further analysis of the mutant, a PCR was performed to amplify the rpsl gene. The amplified DNA then underwent gel electrophoresis to determine if our PCR for the rpsl gene was successful. Unfortunately sequencing software was inaccessible. If sequencing was possible the information could be compared to other rpsl sequences of other organisms through a BLAST search at the NCBI website.
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