Plasmids are extra-chromosomal elements that are normally found in most bacteria. A recombinant plasmid, pGLO™, is used in Escherichia coli for many student experiments, especially as part of the Advanced Placement Biology curriculum. This plasmid contains the green fluorescent protein (GFP) gene under control of the arabinose promoter and regulatory protein, the beta lactamase gene, an origin of replication, and a multiple cloning site. The plasmid is retained in cells due to selective pressure of medium containing ampicillin. Evolutionary fitness is typically considered the reproductive success of a particular genotype/phenotype with emphasis on the particular genetic background of the individual and, hence, plasmid maintenance would seem to be under tight control. Little experimentation has been done on the rate of plasmid loss in bacteria in the absence of selective pressure. In this study, plasmid loss was explored under different selective pressures, especially related to the cost of expression of GFP. This study found strong selective pressure against GFP gene expression in both DH5α and DH10B strains of E. coli, with significantly faster plasmid loss observed in DH10B than in DH5α. There was surprisingly little selective pressure for pGLO plasmid loss when GFP was not actively expressed. Gain of function mutations that allow for positive selection for their occurrence have been well characterized in the scientific literature, however loss of function mutations and subsequent use of negative selection for their retrieval provide a unique opportunity to study another facet of the evolutionary process. This research provides such an example.