The Control of Gene Expression by the Lac Operon

All organisms can adapt to changes in their environment by turning specific metabolic pathways on or off. Jacob and Monod came up with the operon model, which describes how the expression of a gene or group of genes could be turned on or off, as in genome editing, in response to environmental changes, while they were studying enzymes in E. coli bacteria. In particular, they studied β-Galactosidase, an enzyme that is responsible for breaking down the disaccharide lactose into the two monosaccharaides glucose and sugar and therefore allows E. coli to use lactose as an energy source. β-Galactosidase also hydrolyzes lactose to galatose and glucose, promoting the isomerization of lactose to allolactose, and thus delaying the exit of glucose from the acceptor site. The actual gene that is responsible for regulating β-Galactosidase is called the lac operon, which is composed of two major DNA sequences. The first is the promoter/operator sequence responsible for initiating the process of transcription of the structural genes. When this sequence is free, the structural genes will manufacture the β-Galactosidase enzyme. The second sequence is a series of three structural genes involved with lactose metabolism, which are only read when the promoter/operator sequence is unblocked. The regulator gene works with the lac operon, producing a protein called a repressor. This repressor will bind to the promoter/operator sequence of the lac operon and block it in the absence of lactose, thus the structural genes will not be read, and β-Galactosidase is not produced. When lactose is present, it will bind to the repressor protein and inactivate it. This allows the promoter/operator sequence to be free to transcribe the structural genes necessary for making β-Galactosidase. The repressor in E. coli is a protein that attaches to widely spaced sites along a genome and forces the DNA into a loop. These loops imply that there are factors other than DNA and repressors involved in gene control.

Working from a DNA extraction lab, the idea that genes can be switched on and off originated from the theory of allosteric transitions, in which the repressor regulates cellular events by adopting two distinct conformations. One conformation binds with high affinity to the operator and another has a high affinity for the inducer. The purpose of conducting this experiment is to observe the repression and derepression of the lac operon of E. coli. The experimenter hypothesizes that β-Galactosidase will be produced in the bacterial cultures grown in media containing lactose and lactose + glucose and not produced in the culture grown in media containing only glucose. Additionally, the bacterial culture grown in media containing only lactose will produce the most β-Galactosidase.

The materials required for this procedure are E. coli bacterial cultures grown in media containing lactose, glucose + lactose, O-NPG, pipettes, test tubes, test tube rack, and an incubator. Twenty-four to thirty-sex hours before conducting the experiment, the three types of bacterial growth media need to be inoculated with E. coli bacteria and incubated at 37 °C. To begin the experiment, the experimenter obtained three test tubes and labeled one “glucose,” another “lactose,” and the final one “glucose + lactose.” Next, the experimenter transferred twelve drops of E. coli from each medium into each properly labeled test tube with a pipette. Then, 10 drops of O-NPG were added to each tube and mixed thoroughly with the cultures. O-NPG is an indicator and is broken down by β-Galactosidase into a yellow byproduct. Afterwards, the test tubes were placed in the incubator set to 37 °C. The experimenter then checked for color changes every ten minutes for thirty minutes and recorded their observations in the data table. The presence of color was indicated with symbols: ++ for strongly yellow, + for weakly yellow, and – for not yellow.