Difference between revisions of "Part:BBa K5321010"
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====Protease Activity Verification==== | ====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 | + | 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. |
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| − | <img src="https://static.igem.wiki/teams/5321/result/ | + | <img src="https://static.igem.wiki/teams/5321/result/protease-activity-verification-1.jpg" width="800" height="auto"/> |
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| − | '''Figure | + | '''Figure 2 | Enzymatic activity assay of TEV protease extracted from inclusion bodies.''' The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate, under two different reaction buffer conditions (Tris-HCl: 20 mM Tris-HCI+10 mM NaCl+10 mM KCI+1 mM DTT and HEPES: 20 mM HEPES+10 mM NaCI+10 mM KCI+1 mM DTT). The top band corresponds to the intact substrate (tGFP-tevS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (tGFP and tYFP), which increase over time. Samples were taken at 0, 5, 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. | |
Revision as of 03:06, 22 September 2024
GFP_truncated_linker(TEV)_YFP_truncated
Contents
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 148
Illegal AgeI site found at 159 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
We used GFP_truncated_linker(TEV)_YFP_truncated as a cleavage substrate for Plum pox virus protease (TEVp), which is formed by a TEVp cleavage site (ENLYFQS) with truncated Green fluorescent protein (GFP) and the truncated Yellow fluorescent protein (YFP) at both ends, and there is a linker between each of the two, consisting of G and S. When it is cleaved by TEVp, two bands of different sizes can be observed by SDS-PAGE electrophoresis.
Figure 1 | Schematic of GFP_truncated_linker(TEV)_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. The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate, under two different reaction buffer conditions (Tris-HCl: 20 mM Tris-HCI+10 mM NaCl+10 mM KCI+1 mM DTT and HEPES: 20 mM HEPES+10 mM NaCI+10 mM KCI+1 mM DTT). The top band corresponds to the intact substrate (tGFP-tevS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (tGFP and tYFP), which increase over time. Samples were taken at 0, 5, 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.