Part:BBa_K1025006
Bitter Defender Part （RBS_B0030）
iGEM bitter pressure part is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for providing of selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype. This selection pressure was based on the tryptophan dependent tetracycline antiporter expression which functioned in tetracycline culture condition. It is achieved by cloning E. Coli tetracycline antiporter gene (tetA) downstream of our previously constructed tryptophan biosensor which is controlled by tac promoter between NcoI and BamHI restriction sites in pTrc99A vector. We constructed three bitter pressure part by utilizing three different RBS upstream of tetA gene. We transformed these vectors into three previously engineered E. coli with different tryptophan productivity (unpublished data). The tryptophan productivity of these nine strains (three RBS and three previous productivities) was further confirmed by HPLC after culture in M9YE medium for nearly 25h after 0.1mM IPTG induction.Strains carrying our bitter pressure part with RBS B0030 showed good tryptophan dependent growth property within the first 15h after culture. Further, as the increase of tetracycline, the selection pressure increased and the growth rate of strains decreased.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 618
Illegal NgoMIV site found at 693
Illegal NgoMIV site found at 723
Illegal NgoMIV site found at 943
Illegal NgoMIV site found at 1061
Illegal NgoMIV site found at 1328
Illegal NgoMIV site found at 1523 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1750
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