Part:BBa_K133000:Design
CF-mCherry-RGD-Hisstop
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1207
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 764
Illegal BamHI site found at 1275
Illegal BamHI site found at 2239 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 301
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2274
Design Notes
Chimeric fusion protein, which retains the central, most antigenic segment of H. pylori (hypervariable region) flagellin FlaA, while replacing the N- and C-terminal segment by the FliC flagellin from E. coli, which is responsible for the activation of TLR5 (176 AA from the N-terminal and 99 AA from the C-terminal). Thus, combining the right parts of flagellin of E. coli with that of H. pylori enables activation of innate immunity - a characteristic that flagellin of H. pylori alone does not posess - while presenting the antigen of H. pylori for antibody production.
The protein is marked with mCherry red fluorescent protein.
The part was cloned into pSB1.AK3 without terminator for use in bacterial cells (surface expression).
Source
The composite parts of chimeric protein were amplificated by PCR reaction from E. coli and H. pylori genomic DNA respecively. Red Flourescence Protein was obtaind from previousely designed biobrick of 2007 iGEM team Ljubljana containing Red Flourescence Protein with added nuclear localisation signal and mutated PstI restriction site. RGD-Hisstop was obtained with annealing.