Question

You are engineering a bacterium that can be added to sample of drinking water as a biosensor. It will “light up” by expressing a fluorescent protein in response to bisphenol A (BPA)—used to make plastics—leeched into the water. You’ve decided to design your detection scheme using inspiration from the lac operon, and you mutate the lac repressor to respond to BPA instead of allolactose.

Draw out a diagram of the genetic element for the detection scheme, indicating the promotor, the operator, the fluorescent protein gene and the repressor protein.

You build your bacterium, and find that, while the detection scheme works, sensitivity is simply too poor to detect low concentrations of BPA that might still be harmful.

Describe why a signaling cascade, like a kinase cascade or a protease cascade, could be used to improve your sensitivity.

In order to implement a cascade, your repressor can no longer be the direct sensor for BPA. You instead develop a BPA-binding transmembrane protein that localizes to the cell membrane.

Choose either a phosphorylation or a protease cascade. Draw a cartoon scheme that links extracellular BPA detection at the cell membrane to expression of your fluorescent protein. Include three intermediate enzymes (“protease/kinase A”, “protease/kinase B”, “protease/kinase C”) in your cascade.

How could your repressor become inactivated—turning on gene expression—at the end of your cascade (kinase or protease)?

Answer 1

In absence of BPA, membrane receptor would not be bound thus remain inactivated and so the cascade will not function. The repressor protein produced will bind to the operator region and shield the promotor from RNA polymerase and transcription and translation of the fluorescent protein will not take place.