Difference between revisions of "Team:CU-Boulder/Isolation"

Bacterial microcompartments are protein-based organelles that can enclose enzymes and other proteins, however without successful isolation of the compartments they do not have much use. We used methods previously described [1] to isolate the microcompartments through lysis and ultracentrifugation then confirmed isolation of the micro compartments in two ways:

1. Fluorescent foci (Fusion Red-LVA and Fusion Red Luciferase LVA) to identify the compartments in fluorescent imaging. With Eut-C tags, the fluorescent markers accumulate inside the compartment, allowing them to be clearly visible inside the compartment. The LVA protein degradation tag targets any excess fluorescent proteins for degradation to allow a sufficient background for viewing the compartments in vivo.

2. Electron Microscopy with various radiological stains on a copper grid. We found that flash freezing the compartments in liquid ethane before viewing with electron microscopy preserved the integrity of the compartment.