Difference between revisions of "Part:BBa K4632002:Design"
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| + | C.What we have done?（SCAU-China 2023） | ||
| + | In our design, we aimed to introduce a gene fragment encoding an active Cry3A-like toxin (66.6 kDa) into Escherichia coli using the pET30a vector to confer it with the ability to produce an active Cry3A-like toxin. | ||
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| + | To achieve secretion expression, we added a signal peptide sequence, OmpA, to the N-terminus of Cry3A-like toxin. This was done to direct the transport of Cry3A-like toxin to the extracellular space. OmpA is a well-established signal peptide in Escherichia coli for the secretion expression of foreign proteins. Our SDS-PAGE results confirmed the successful secretion expression of Cry3A-like toxin. | ||
| + | |||
| + | Furthermore, we fused a 6×His tag to the C-terminus of Cry3A-like toxin to facilitate subsequent protein purification and Western blot-specific characterization experiments. | ||
| + | |||
| + | In addition, to validate the toxicity of the designed Cry3A-like toxin against red imported fire ants, we selected homologous receptors of Cry3A-like toxin known from NCBI in red imported fire ants. We then conducted molecular docking studies to assess the protein-protein interaction capability of Cry3A-like toxin, thus evaluating its toxic effects. Detailed results are presented in the characterization section. | ||
Revision as of 13:45, 8 October 2023
C.What we have done?（SCAU-China 2023）
In our design, we aimed to introduce a gene fragment encoding an active Cry3A-like toxin (66.6 kDa) into Escherichia coli using the pET30a vector to confer it with the ability to produce an active Cry3A-like toxin.
To achieve secretion expression, we added a signal peptide sequence, OmpA, to the N-terminus of Cry3A-like toxin. This was done to direct the transport of Cry3A-like toxin to the extracellular space. OmpA is a well-established signal peptide in Escherichia coli for the secretion expression of foreign proteins. Our SDS-PAGE results confirmed the successful secretion expression of Cry3A-like toxin.
Furthermore, we fused a 6×His tag to the C-terminus of Cry3A-like toxin to facilitate subsequent protein purification and Western blot-specific characterization experiments.
In addition, to validate the toxicity of the designed Cry3A-like toxin against red imported fire ants, we selected homologous receptors of Cry3A-like toxin known from NCBI in red imported fire ants. We then conducted molecular docking studies to assess the protein-protein interaction capability of Cry3A-like toxin, thus evaluating its toxic effects. Detailed results are presented in the characterization section.