Difference between revisions of "Team:CU-Boulder/Isolation"
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accumulate inside the compartment, allowing them to be clearly visible inside the | accumulate inside the compartment, allowing them to be clearly visible inside the | ||
compartment. The LVA protein degradation tag targets any excess fluorescent proteins | compartment. The LVA protein degradation tag targets any excess fluorescent proteins | ||
| − | for degradation to allow a sufficient background for viewing the compartments in vivo.</p> | + | for degradation to allow a sufficient background for viewing the compartments in vivo. </br></br> |
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| + | 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.</p> | ||
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Latest revision as of 03:40, 2 November 2017
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
