Composite

Part:BBa_K1025007

Designed by: Weifan Liang   Group: iGEM13_Tsinghua-E   (2013-09-15)
Revision as of 06:53, 15 September 2013 by Registry (Talk | contribs)

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Bitter Defender Part（RBS_B0032）

iGEM bitter pressure part(B0032) 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 We transformed these vectors into three previously engineered E. coli with different tryptophan productivity (unpublished data).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 B0032 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. However, with increased selection pressure,it could be observed that with time window of about 15h, strains with higher tryptophan overproduction showed much higher growth rate with the growth of tryptophan non producer trp000 nearly inhibited by high concentration of tetracycline.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 616
    Illegal NgoMIV site found at 691
    Illegal NgoMIV site found at 721
    Illegal NgoMIV site found at 941
    Illegal NgoMIV site found at 1059
    Illegal NgoMIV site found at 1326
    Illegal NgoMIV site found at 1521
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1748


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