Authors:

Soren Spina

Date Created:

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About Paper:

Phospholipase C (PLC) is essential to study due to its ability to generate second messengers IP3 (inositol triphosphate) and DAG (diacylglycerol) through its hydrolysis of the membrane lipid phosphatidylinositol 4,5- bisphosphate (PIP2). IP3 triggers Ca2+release, which is important for normal cardiac contractility, while DAG production activates protein kinase C, causing downstream expression of pro-inflammatory genes. Thus, PLC signaling is directly implicated in cardiovascular disease and many other health complications. We are working to detect DAG produced in the membrane, as its presence is directly proportional to PLC activity. We generated a construct to express a DAG biosensor, consisting of the rat PKCδ C1 domain tagged with GFP. Fluorescent measurements of DAG production in the presence of purified PLC can allow for direct qualitative and quantitative conclusions about PLC activity. We are generating this biosensor by utilizing rigorous cloning, transformation, and protein purification techniques. The biosensor is being expressed in E. coli due to the versatility, quick growing times, and decreased cost of bacterial cells compared to eukaryotic cells. Our biosensor has been successfully cloned and sequenced, and next steps are to optimize protein expression conditions, purify the biosensor, and test the biosensor in a model membrane system using total internal reflection fluorescence microscopy. With this biosensor, research involving PLC and similar DAG-induced systems will have more easily interpretable data, allowing it to be used as an impactful tool in research for years to come.

Source:

Purdue University / 2023

Topics:

No topics listed

Co-authors:

Soren Spina