Difference between revisions of "Part:BBa K5321013"

 
Line 1: Line 1:
 +
<partinfo>BBa_K5321013 short</partinfo> 
 +
<br>
 +
===Sequence and Features===
 +
<partinfo>BBa_K5321013 SequenceAndFeatures</partinfo>
  
−
__NOTOC__
+
===Usage and Biology===
−
<partinfo>BBa_K5321013 short</partinfo>
+
  
−
666
+
We used MBP_linker(TEV)_GFP_truncated_linker(PPV)_YFP_truncated as a cleavage substrate for Tobacco Etch Virus protease (TEVp), which is formed by a TEVp cleavage site (ENLYFQS) between MBP soluble tag and truncated Green fluorescent protein (GFP)-truncated Yellow fluorescent protein (YFP) fusion protein. When it is cleaved by TEVp, two bands of different sizes can be observed by SDS-PAGE electrophoresis.Also, there is a PPVp leavage site between the truncated GFP and YFP, which can be used to test the activity of PPVp as well.
  
−
<!-- Add more about the biology of this part here
+
<html>
−
===Usage and Biology===
+
<div align="center">
 +
        <img src="https://static.igem.wiki/teams/5321/parts/mbp-g-y.png" width="1000px" height="auto"/>
 +
</div>
 +
</html><br>
 +
'''Figure 1 | Schematic of MBP_linker(TEV)_GFP_truncated_linker(PPV)_YFP_truncated.'''<br>
  
−
<!-- -->
+
===Characterization===
−
<span class='h3bb'>Sequence and Features</span>
+
====Protease Activity Verification====
−
<partinfo>BBa_K5321013 SequenceAndFeatures</partinfo>
+
Since our system relies on the protease both amplifying the signal and triggering the release of the final colloidal gold output, it is crucial to verify the target protease activity to ensure that the enzymes used in our experiments are active and functioning as expected. To achieve this, we designed a experiment to verify the enzyme activity under controlled conditions (you can find more detailed information about this experiment in our protocol). We validated the activity of two intact proteases and one split protease. For the intact TEV proteases, we mixed a calculated amount of the enzyme with its corresponding substrate and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were taken at different time points. The reaction was stopped with SDS loading buffer, followed by electrophoresis. Enzyme activity was confirmed by observing the reduction in substrate and the presence of cleavage product bands.
 +
 
 +
<html>
 +
<div align="center">
 +
        <img src="https://static.igem.wiki/teams/5321/result/tev-for-mbp-g-y-results.png" width="800" height="auto"/>
 +
</div>
 +
</html>
  
 +
'''Figure 2 | Enzymatic activity assay of TEV protease extracted from inclusion bodies.''' MW: MBP-tevS-G-Y: 65.6kDa  G-Y: 22.6kDa    MBP:43.1kDa. The top band corresponds to the intact substrate (MBP-tevS-tGFP-ppvS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products, which increase over time. Samples were taken at 0, 10, 30, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes.
  
−
<!-- Uncomment this to enable Functional Parameter display
+
The results confirm that the TEV protease extracted from inclusion bodies is active.
−
===Functional Parameters===
+
−
<partinfo>BBa_K5321013 parameters</partinfo>
+
−
<!-- -->
+

Revision as of 07:48, 22 September 2024

MBP_GFP_truncated_linker(TEV)_YFP_truncated

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 402
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1330
    Illegal AgeI site found at 1341
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 100

Usage and Biology

We used MBP_linker(TEV)_GFP_truncated_linker(PPV)_YFP_truncated as a cleavage substrate for Tobacco Etch Virus protease (TEVp), which is formed by a TEVp cleavage site (ENLYFQS) between MBP soluble tag and truncated Green fluorescent protein (GFP)-truncated Yellow fluorescent protein (YFP) fusion protein. When it is cleaved by TEVp, two bands of different sizes can be observed by SDS-PAGE electrophoresis.Also, there is a PPVp leavage site between the truncated GFP and YFP, which can be used to test the activity of PPVp as well.


Figure 1 | Schematic of MBP_linker(TEV)_GFP_truncated_linker(PPV)_YFP_truncated.

Characterization

Protease Activity Verification

Since our system relies on the protease both amplifying the signal and triggering the release of the final colloidal gold output, it is crucial to verify the target protease activity to ensure that the enzymes used in our experiments are active and functioning as expected. To achieve this, we designed a experiment to verify the enzyme activity under controlled conditions (you can find more detailed information about this experiment in our protocol). We validated the activity of two intact proteases and one split protease. For the intact TEV proteases, we mixed a calculated amount of the enzyme with its corresponding substrate and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were taken at different time points. The reaction was stopped with SDS loading buffer, followed by electrophoresis. Enzyme activity was confirmed by observing the reduction in substrate and the presence of cleavage product bands.

Figure 2 | Enzymatic activity assay of TEV protease extracted from inclusion bodies. MW: MBP-tevS-G-Y: 65.6kDa G-Y: 22.6kDa MBP:43.1kDa. The top band corresponds to the intact substrate (MBP-tevS-tGFP-ppvS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products, which increase over time. Samples were taken at 0, 10, 30, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes.

The results confirm that the TEV protease extracted from inclusion bodies is active.