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Isolation of Drug Resistant Mutants

Background & Introduction 

The metabolic and structural natures of all organisms are determined by the genetic information within the DNA molecule(s) of the cell.  The complete genetic information of an organism, or genome, is divided into segments, called genes.  A gene is a linear sequence of nucleotides that forms a functional unit of the chromosome or plasmid.  Many genes code for the synthesis of RNA and proteins, thereby determining the sequences of ribonucleotide bases and amino acids, respectively.  The faithful replication of DNA ensures passage of the proper hereditary information from parent cell to daughter cells.  However, genomes of organisms are subject to change by a variety of mechanisms.  In bacteria, heritable changes in the DNA sequence can result from recombination events (facilitated by transformation, conjugation and transduction), and by mutations.  Mutation is defined as a permanent, heritable change in the genetic material.  Mutations usually change the sequence of nucleotides in DNA, which ultimately leads to changes in RNA and protein macromolecules.  

Spontaneous mutations occur randomly at a very low rate due to infrequent errors made by DNA polymerase during DNA replication.  In most bacterial populations the mutations occur at a rate of approximately one per 10 million (107) to 10 billion (1010) organisms.  Although a mutation in a particular gene is a rare event, mutations can be observed in bacterial systems because bacteria reproduce so rapidly and yield quite large populations (~1-3 x 109 cells/mL) over a short period of time.  Therefore, a bacterial culture grown to a high titer will always contain a few mutants.  Some mutations may result in loss of the ability to degrade sugars (ex., lactose) or to synthesize key nutrients (ex., amino acids), some may give rise to antibiotic resistance, and some may result in no detectable change.  Mutant bacteria usually do not grow as well as the wild-type bacteria because most changes are harmful, or at least not helpful.  If, however, conditions change in the environment and favor a mutant cell, it will be able to out-compete and outgrow the cells that do not have the advantageous mutation.  

In this exercise, we will select for the growth of bacteria that are resistant to the antibiotic streptomycin.  As was discussed earlier, antibiotics kill or inhibit bacteria by interfering with essential processes such as protein synthesis, cell wall synthesis, and DNA replication.  Streptomycin kills bacteria by binding to the bacterial 70S ribosome to prevent protein synthesis.  (Note: It does not stop protein synthesis in eucaryotic cells because it doesn't bind to eucaryotic 80S ribosomes).  Escherichia coli that are sensitive to streptomycin can become resistant with just one mutation.  This mutation alters the ribosome so that streptomycin can no longer bind to it, however the mutation does not alter the normal function of the ribosome.  In the presence of streptomycin, bacteria that have this mutation will most likely survive and grow, whereas those without the mutation will die.  After a few generations, most survivors will be resistant to the streptomycin.  It is important to note that antibiotics do not induce mutations, but they can create environments that favor the survival of mutant resistant organisms. 

Throughout this experiment it is important to keep in mind that the events we demonstrate in vitro ("in glass" or in a test tube) may also occur in a patient taking antibiotics.  For example, a patient suffering from gonorrhea, a sexually transmitted disease, may have billions of Neisseria gonorrheae in his or her body.  If one or more of these bacteria spontaneously develop resistance to an antibiotic such as streptomycin, and streptomycin is administered to fight the infection, then resistant mutants are selected during the antibiotic therapy.  Therefore, it is extremely important to identify the antibiotic to which an organism is most sensitive before using any antibiotic to treat a disease. 

Procedure

Be sure to watch both the Streptomycin Resistance isolation and confirmation videos

Results for initial mutant selection and confirmation:

Pour plates with and without streptomycin

Broth tubes with and without streptomycin

Confirmation TSA plate with broth and plate innoculants

Confirmation TSA+streptomycin plate with broth and plate innoculants

Pour plates with and without streptomycinBroth tubes with and without streptomycin
TSA without streptomycinTSA with streptomycin

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