Difference between revisions of "Part:BBa K1025007"

 
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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 We transformed these vectors into three previously engineered E. coli with different tryptophan productivity (unpublished data).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.
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iGEM bitter pressure part(RBS_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. It is almost the same with iGEM bitter pressure part(RBS_B0030)（[https://parts.igem.org/Part:BBa_K1025006 BBa_K1025006]） 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（[https://parts.igem.org/Part:BBa_K1025005 BBa_K1025005]） 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_K1025007 parameters</partinfo>
 
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<span class='h3bb'>Sequence and Features</span>
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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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<partinfo>BBa_K1025007 SequenceAndFeatures</partinfo>
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[[File:mut.jpg|375px|thumb|left]]
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[[File:-2.png|455px|thumb|left]]
  
  
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===Functional Parameters===
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1025007 SequenceAndFeatures</partinfo>

Revision as of 17:05, 18 September 2013

Bitter Defender Part（RBS_B0032）

iGEM bitter pressure part(RBS_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. It is almost the same with iGEM bitter pressure part(RBS_B0030)（BBa_K1025006） 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（BBa_K1025005） 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 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