Molecular cloning is used to study the relationship between the structure of a protein and its function. However, the process can be time consuming and not necessarily practical in the constraints of a 3hr weekly lab. This “dry” lab walks students through key points of the process used to develop the plasmids needed to express altered ZO-1 proteins within epithelial cells in order to understand the role of each binding domain in the formation and function of the tight junction. Students practice finding and labeling protein domains and enzyme restriction sites within the ZO-1 DNA sequence and plasmid sequences. They also explore some methods used to put a sequence of interest into expression vectors. This lab can be used as a stand-alone unit or as the final exercise of your favorite cloning wet lab. This lab is recommended for a 300 or 400 level cell and molecular biology type course.
Keywords: protein structure, cloning, sequencing, protein structure-function, tight junctions
University of Ottawa (2019)
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