Difference between revisions of "Part:BBa K1025006"

 
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<partinfo>BBa_K1025006 short</partinfo>
 
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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 We transformed these vectors into three previously engineered E. coli with different tryptophan productivity (unpublished data).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.
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iGEM bitter pressure part(RBS_B0030) 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. It is almost the same with iGEM bitter pressure part(RBS_B0032) except for the second RBS.
  
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===Usage and Biology===
 
===Usage and Biology===
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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).
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===Functional Parameters===
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<partinfo>BBa_K1025006 parameters</partinfo>
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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.
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[[File:mut.jpg|375px|thumb|left]]
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[[File:-2.png|455px|thumb|left]]
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===Reference===
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===Functional Parameters===
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[1] Schaaper, R. M. MECHANISMS OF MUTAGENESIS IN THE ESCHERICHIA-COLI MUTATOR MUTD5 - ROLE OF DNA MISMATCH REPAIR. Proc. Natl. Acad. Sci. U. S. A.85, 8126-8130, doi:10.1073/pnas.85.21.8126 (1988).
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Revision as of 16:41, 18 September 2013

_ Bitter Defender Part （RBS_B0030）

iGEM bitter pressure part(RBS_B0030) 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. It is almost the same with iGEM bitter pressure part(RBS_B0032) except for the second RBS.

Usage and Biology

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).

Functional Parameters

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.

File:Mut.jpg
375px
File:-2.png
455px


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 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1750


Reference

[1] Schaaper, R. M. MECHANISMS OF MUTAGENESIS IN THE ESCHERICHIA-COLI MUTATOR MUTD5 - ROLE OF DNA MISMATCH REPAIR. Proc. Natl. Acad. Sci. U. S. A.85, 8126-8130, doi:10.1073/pnas.85.21.8126 (1988).