Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

The aim of this procedure is to acquire time-lapse fluorescence images of growing e coli colonies, expressing co track constructs to identify the number of newly produced protein molecules. This is accomplished by first preparing a sample of cells, coex expressing a protein of interest fused to a fluorescent Cora reporter, and a protease that specifically cleaves between the Cora reporter and the protein of interest. The second step is to prepare a gel of low melting temperature Aris made with M nine minimal media.

Next, an AROS gel pad is prepared for use with a temperature controlled sample chamber. The e coli cells are placed on the gel and the chamber is assembled. The final step is to place the chamber on a fluorescence microscope and image several growing cell colonies at regular intervals over multiple generations.