Antibiotic is a general term that describes a substance, such as a medicine, that kills or slows the growth of microbes (National Institute of Allergy and Infectious Diseases, 2009). One example of an antibiotic is Streptomycin. Streptomycin is derived from the bacterium Streptomyces griseus and is a member of the aminoglycoside family (Voet and Voet, 2004). Streptomycin works as a protein synthesis inhibitor. By binding to the 30S subunit of the bacterial ribosome and interfering with tRNA binding capabilities, it leads to codon misreading. This in turn leads to protein synthesis inhibition, and ultimately cell death.
Streptomycin was first discovered in 1943 by a graduate student in a laboratory at Rutgers University. Since then, it has been used to treat a number of infectious diseases. Streptomycin also has implications in veterinary medicine, where it is often used to treat gram negative bacteria in large animals. Most notably, Streptomycin was the first antibiotic that was found to cure Tuberculosis.
However, mutations in the genotype of an organism can confer resistance to antibiotics such as Streptomycin. In the case of the bacteria Escherichia coli, a mutation in the rpsL gene, which codes for the rpS12 ribosomal protein to which Streptomycin binds, can result in E. coli resistance to the antibiotic (Sharma et al., 2007). Due to this type of spontaneous mutation, Streptomycin loses its affinity for ribosomal binding, and is therefore rendered ineffective. Consequently, protein synthesis remains unaltered, and the cell is able to grow and reproduce normally.
In this experiment, we isolated different Streptomycin mutants of the E. coli bacteria in order to estimate the frequency of mutations that occurred in the rpsL gene. The mutants were tested and cloned in order to determine which of the three resultant phenotypes (Streptomycin resistant, Streptomycin intermediate, or Streptomycin dependent) were acquired by the mutation. The rpsL gene of E. coli was then PCR amplified in order to allow for sequencing by Genewiz. The sequence was then further analyzed using bioinformatics tools.
