Part:BBa_K5292053:Design
Mtac415
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
During the detailed design of the Mtac promoter, we considered the following: Target mutation region: We focused on mutating the 16 bp sequence between the -35 and -10 regions of the tac promoter, a critical site for RNA polymerase binding. Mutations in this region could significantly impact promoter strength, and choosing this area for random mutagenesis allows us to explore how various mutations affect promoter performance. Mutation diversity: By designing degenerate primers, we ensured the generation of a highly diverse library of promoter variants. This diversity is crucial for selecting mutants that might enhance ICCG expression in downstream applications. Seamless cloning method: To prevent the introduction of unwanted sequences or mutations during the cloning process, we opted for seamless cloning, which helps maintain the structural integrity of the promoter and reduces experimental errors. Precision in expression regulation: We used the GFP fusion protein to characterize the promoter strength of different mutants. This approach provides a direct and quantitative way to measure promoter activity, offering reliable data for model construction and optimization in the future.
Source
The mutated tac promoter is derived from an artificial construct, originally engineered by combining elements from the trp and lac promoters in E. coli.