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

 
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not have much use. We used methods previously described [1] to isolate the
 
not have much use. We used methods previously described [1] to isolate the
 
microcompartments through lysis and ultracentrifugation then confirmed isolation of the
 
microcompartments through lysis and ultracentrifugation then confirmed isolation of the
−
micro compartments in two ways:
+
micro compartments in two ways: </br></br>
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 +
 
 
1. Fluorescent foci (Fusion Red-LVA and Fusion Red Luciferase LVA) to identify the
 
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
 
compartments in fluorescent imaging. With Eut-C tags, the fluorescent markers
 
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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<img src = "https://static.igem.org/mediawiki/2017/d/dd/T-CU_Boulder--IsolationResultOne.jpeg"/>
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<p><i>Figure 1: EutS compartment containing fusion red </br> tagged with Eut C
 +
2. Electron Microscopy with various </br> radiological stains on a copper grid.</i></p>
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<img src = "https://static.igem.org/mediawiki/2017/1/18/T-CU_Boulder--IsolationResultTwo.jpeg"/>
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<p><i>Figure 2: EutS compartment with negative stain</i></p>
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</sectionTwo>
 
</sectionTwo>

Latest revision as of 03:40, 2 November 2017

• 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