Team:WLC-Milwaukee/Description
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Description
Instruction on gene regulation is ubiquitous in every biology class. Although it is an essential topic, the quality of educational resources available is poor. This is to no fault of the educator; gene regulation is difficult to explain verbally. We seek to improve the quality of gene regulation instruction by creating a hands-on lab kit that communicates molecular processes through color changes.
To do this, we designed a lab kit and accompanying manual that allows students to visualize gene regulation through the regulatory components of the lac operon, a system of genes responsible for converting lactose to glucose. We chose to use this because it is most frequently used to teach gene regulation in current biology curricula. Additionally, the regulatory components of the lac operon are well-documented and understood.
What are operons?
In prokaryotes, multiple genes that are grouped close together on a chromosome, contribute to the same metabolic pathway, and are under the control of one promoter are called operons. The benefit of being under the control of one promoter is that every gene can be expressed at once. An operon may have regulatory components that repress or activate gene expression depending on the environment. The lac operon’s genes are activated by the presence of allolactose, a byproduct of lactose degradation.
The lac operon contains a gene (lacZ) that expresses b-galactosidase. B-galactosidase converts lactose into glucose, which the cell can metabolize to create usable energy. Downstream of lacZ is the gene lacY which expresses lac permease. Lac permease creates channels in the membrane of the E. coli cell that allow lactose to enter. lacZ and lacY are referred to as structural genes.
The first regulatory component of the lac operon is lacI. lacI expresses the lac repressor protein constantly. The lac repressor protein binds to a regulatory gene called lacO. This prevents the expression of the structural genes by blocking DNA polymerase. Allolactose, a disaccharide produced from lactose metabolism, binds to the lac repressor protein and changes its shape, causing it to dissociate from lacO. To summarize, when lactose concentration is high, the lac repressor protein dissociates from lacO which allows the expression of the structural genes.
The second regulatory component of the lac operon is the CAP binding site. When the cell is running out of glucose, the molecule cAMP becomes plentiful. cAMP binds with the molecule CAP, and the cAMP/CAP complex binds to the CAP binding site. This binding increases the expression of the structural genes lacZ and lacY. When allolactose is bound to the lac repressor protein and is dissociated from lacO, the cAMP/CAP complex binding to the CAP binding site accelerates expression. Therefore, when glucose concentration is low (cAMP concentration is high), the expression of lacZ and lacY is amplified. Lactose must still be present for lacZ and lacY to be expressed at all, but the absence of glucose significantly increases expression.
Our Project
Our project combines the regulatory components of the lac operon with the gene bla. The bla gene expresses b-lactamase which turns red in the presence of the substrate nitrocefin. Therefore, when conditions favor structural gene expression, a culture of E. coli with our designed plasmid should turn red when exposed to nitrocefin. By mutating the regulatory components and observing the subsequent color change or lack thereof, we hope to encourage an intimate understanding of gene regulation.
Sources
- Brooker, R. (2015). Genetics: Analysis and Principles (5th ed.). New York, New York: McGraw-Hill Education.
- Mader, S. S., & Michael, W. (2013). Biology (11th ed.). New York, New York: McGraw-Hill Education.
