Team:CU-Boulder/Isolation

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• Isolation •

Compartment 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.

Figure 1: EutS compartment containing fusion red
tagged with Eut C 2. Electron Microscopy with various
radiological stains on a copper grid.

Figure 2: EutS compartment with negative stain