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The Escherichia coli lac Operon is controlled by a regulatory system that has been the subject of intensive study for the past fifty years. The system creates metabolic efficiency by responding to the levels of environmental lactose. In the absence of lactose, the LacI protein acts as a repressor of transcription from the lac promoter. Transcription begins when lactose binds to LacI, which results in the expression of three genes involved in lactose uptake and catabolism. The lac promoter is the most commonly used promoter in the field of synthetic biology. Although it is widely used, the lac promoter is known to have leaky transcription, meaning that transcription takes place even when the repressor is present and the inducer is absent. In an effort to redesign the lac promoter, we tested pLac variants that were reported to have a higher affinity for RNA polymerase than the wild-type. We also compared three mutants of the LacI repressor that were reported to have increasing affinity for the pLac promoter. Using GFP reporter constructs, we found that the pLacIQ1 promoter showed much higher levels of transcription than the wild-type promoter. Of the twelve combinations of promoters and repressors tested in the presence and absence of an inducer, we discovered that the wild-type LacI repressor protein with the pLacIQ1 mutant promoter is the best combination for high levels of induction and low levels of leaky transcription. Our results promise to help synthetic biologists design and build systems with tighter regulatory control.
Butylated hydroxyanisole (BHA) is a widely used food preservative. It is an antioxidant that reacts with oxygen free radicals, and thus, slows down the oxidation process of certain food components that can alter taste or color. When BHA is ingested, it is absorbed through the gastrointestinal tract and metabolized. BHA has been shown to have both positive and negative effects on the body. By adding various concentrations of BHA to rat hepatocyte cultures, the release of cytochrome c was monitored to determine the magnitude of BHA that causes apoptosis of the hepatocyte cells. The subacute magnitudes of BHA were administered to the hepatocyte cultures for various durations of time to determine their effect. This gave an optimal magnitude and duration of subacute levels of BHA in order to reduce the release of cytochrome c that leads to hepatocyte apoptosis.
A number of genomes use genetic codes that are different from the standard genetic code. Since many of these genomes have recently been sequenced, we can now study the evolution of the genetic codes by examining the amino acid frequencies encoded by these genomes. We calculated the amino acid frequencies encoded by twenty-two genomes using codon usage frequencies tabulated from GenBank. By chi-square analysis, no significant differences were observed in the amino acid frequencies encoded by fourteen different genomes that use the standard genetic code. However, among eight genomes that employ nonstandard genetic codes, we found differences between three animal mitochondrial genomes (Fasciola hepatica, Drosophila melanogaster, and Homo sapiens) and the remaining 19 genomes. Of the genomes that were studied, we also observed a trend between the usage of an amino acid and its occurrence in the genetic code. Taken together, our study supports the hypothesis that the standard genetic code formed early in the development of modern life and evolved in different lineages to form several nonstandard genetic codes.