Difference between revisions of "Part:BBa K5382130"

Latest revision as of 14:20, 1 October 2024

CL7-linker-sfGFP_Green fluorescent protein and CL7 complex

The CL7 is a protein tag engineered from colicin CE7. In E.coli, CE7 is a bacteriocin that belongs to the colicin DNase family and has endonuclease activity. Through engineering, CL7 removed DNA binding and catalytic activity, but retained its high affinity binding ability to the corresponding inhibitory protein Im7. This property makes CL7 a useful tool for protein purification and molecular biology applications.
The linker here is used to connect CL7 and sfGFP short peptide sequence.
The sfGFP, namely the super folding green fluorescent protein (Superfolder GFP), is a genetically engineered green fluorescent protein (GFP) variations.
In the experimental design, we took advantage of the high affinity between CL7 and Im7 and incubated CL7-Linker-sfGFP to bind to Inak-linker-Im7(another composite part we registered), thus anchoring the integrated system to the membrane surface. Finally, the Inak-linker-Im7-CL7-linker-sfGFP system was demonstrated on the membrane surface(Figure 1), and the function of sfGFP protein was tested by double fluorescence verification (for details, refer to the engineering part in our wiki) to see whether the system was successfully displayed on the membrane surface.
It should be noted that the system we designed uses the high affinity of CL7 and Im7, so some components can be flexibly replaced according to needs, and are suitable for different use scenarios according to different needs. For example, we can according to different cells to replace the ice nucleated protein, or according to different targeting targets to replace sfGFP with different single-chain antibodies, nano antibodies and so on.

Figure 1. Schematic diagram of the targeting plug-in pairing system for Im7 protein to CL7 fusion pair displayed on the EcN surface.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 158
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 158
Illegal NheI site found at 1247
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 158
Illegal BglII site found at 106
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 158
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 158
Illegal AgeI site found at 56
Illegal AgeI site found at 70
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K5382130 short</partinfo>
-CL7 is an engineered variant of colicin CE7, which is a protein tag. In Escherichia coli, CE7 is a bacteriocin that belongs to the colicin DNase family and has endonuclease activity. Through engineering, CL7 removed DNA binding and catalytic activity, but retained its high affinity binding ability to the corresponding inhibitory protein Im7. This property makes CL7 a useful tool for protein purification and molecular biology applications. Would here is used to connect CL7 and sfGFP short peptide sequence. SfGFP, namely the super folding green fluorescent protein (Superfolder GFP), is a genetically engineered green fluorescent protein (GFP) variations. Formed by the union of the three series of composite parts, mainly through the CL7 and Im7 high affinity ability, through the incubation makes CL7 - would - sfGFP and Inak - would - Im7 (we register another composite components), and anchor integration system in the membrane surface. Finally, the Inak-linker-Im7-CL7-linker-sfGFP system was displayed on the surface of the membrane, so that sfGFP protein could play a role. Here, the role of sfGFP protein was tested by double fluorescence verification (for details, go to the sequence design part) whether the system was successfully displayed on the membrane surface. It should be noted that the system we designed uses the high affinity of CL7 and Im7, so some components can be flexibly replaced according to needs, and are suitable for different use scenarios according to different needs. For example, according to different cells to replace the ice nucleated protein, according to different targeting targets to replace sfGFP with different single-chain antibodies, nano antibodies and so on.
+The CL7 is a protein tag engineered from colicin CE7. In <i>E.coli</i>, CE7 is a bacteriocin that belongs to the colicin DNase family and has endonuclease activity. Through engineering, CL7 removed DNA binding and catalytic activity, but retained its high affinity binding ability to the corresponding inhibitory protein Im7. This property makes CL7 a useful tool for protein purification and molecular biology applications. <br>
+The linker here is used to connect CL7 and sfGFP short peptide sequence.<br>
+The sfGFP, namely the super folding green fluorescent protein (Superfolder GFP), is a genetically engineered green fluorescent protein (GFP) variations.<br>
+In the experimental design, we took advantage of the high affinity between CL7 and Im7 and incubated CL7-Linker-sfGFP to bind to Inak-linker-Im7(another composite part we registered), thus anchoring the integrated system to the membrane surface. Finally, the Inak-linker-Im7-CL7-linker-sfGFP system was demonstrated on the membrane surface(Figure 1), and the function of sfGFP protein was tested by double fluorescence verification (for details, refer to the engineering part in our wiki) to see whether the system was successfully displayed on the membrane surface.<br>
+It should be noted that the system we designed uses the high affinity of CL7 and Im7, so some components can be flexibly replaced according to needs, and are suitable for different use scenarios according to different needs. For example, we can according to different cells to replace the ice nucleated protein, or according to different targeting targets to replace sfGFP with different single-chain antibodies, nano antibodies and so on.<br>
+https://static.igem.wiki/teams/5382/part-pictures/im7-cl7.png<br>'''Figure 1.''' Schematic diagram of the targeting plug-in pairing system for Im7 protein to CL7 fusion pair displayed on the EcN surface.