Participants in the UCSB Summer Biochemistry Lab Course clone, mutate, purify, and charactere mutant forms of the CheY protein. This protein is an important regulator of bacterial chemotaxis. During the first week, participants sub-clonened the CheY gene from the pCW plasmid into the pET-28b vector in order to create a His-tagged recombinat protein. During the second week, the recombinant protein was purified using Ni-affinity chromatography and mutant forms of CheY were created using PCR-based site-directed mutagenesis technology. During the third week, proteins were expressed, purified and characterized by mass spectrometry. The binding of Flim peptide to CheY was characterized by fluorescence spectrophotometry in order to determine the pH-dependence of the binding constant.
