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>>Name: Yoo D. Moon
>>Name: Siwoo Kim
>>Name: Kwangse lee
>>Name: Hee Lee

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UMBC Biol302L: Genetics Lab

              To obtain spontaneous Streptomycin mutants of E. coli, growth on both TBAB and TBAB+Strep plates were observed. The number of colonies on the TBAB plate was an average of 336.5 and on the TBAB+Strep plate was 1.25. Colonies grown on the Strep plate are mutants with the average mutation frequency 3.44 x 10 ^ -10 Mutants/cfu. The frequency is low due to the proofreading mechanisms in a cell that corrects errors in the sequence of nucleotide during the replication of DNA.

              To test mutants to determine whether they are Strep-dependent of Strep-resistant, mutant colonies on the Strep plate were transferred to a new Strep plate and TBAB plate and incubated overnight. Growth was observed on both Strep and TBAB plates and this identifies that the mutants are Strep-resistant. If the growth occurred only on Strep plate, the mutants would have been Strep-dependent because the strain needs the presence Streptomycin to survive.

              The types of mutation that can result in these phenotypes is a missense mutation in which a foreign amino acid is formed by a base pair mutation. If a nonsense mutation or frame shift was occurred, no growth would have been observed on both plates because no functional ribosome would have made by knocked out genes. The Strep-resistant colonies appeared because rpS12 protein was altered to prevent Streptomycin binding and this indicates the types of mutation can not be neutral or silent mutation.

              The rpsL gene of the mutant was amplified by a PCR and the sequence of it was analyzed using a BLAST tool available at NCBI. Comparison of rpsL gene sequences of our members shows that mutations occurred at position of 72, 181, 265, 325, and 353. The sequence of each Strep-resistant mutant differs from one amino acid at multiple different positions.