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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> |
+ | |||
+ | 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> | ||
</div> | </div> | ||
</div> | </div> |
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.
![](https://static.igem.org/mediawiki/2017/d/dd/T-CU_Boulder--IsolationResultOne.jpeg)
Figure 1: EutS compartment containing fusion red tagged with Eut C 2. Electron Microscopy with various radiological stains on a copper grid.
![](https://static.igem.org/mediawiki/2017/1/18/T-CU_Boulder--IsolationResultTwo.jpeg)
Figure 2: EutS compartment with negative stain