Coding

Part:BBa_K2433002

Designed by: Tamar Av-Shalom   Group: iGEM17_British_Columbia   (2017-10-25)
Revision as of 05:13, 1 November 2017 by Emmastanley (Talk | contribs)


ietAS

ietAS is composite part made up of ietA an ietS, the genes encoding the toxin antitoxin system ietAS native to tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. This part comes from the nopaline type Ti plasmid pTiC58.The ietAS operon was given its name after the incompatibility enhancer of the Ti plasmid. ietAS has been shown in literature to be important in reducing the transconjugant efficiency for different incoming Ti plasmids as well as contributing to the stability of plasmids harboring this construct.


ietAS is a type II toxin/antitoxin system, meaning both ietA and ietS are proteins. In ietAS, the antitoxin ietA is upstream of the toxin ietS. This is typical of type II toxin/antitoxin systems. ietA is less stable, but expressed at higher levels. ietS is more stable, and expressed at lower levels.


ietA neutralizes the effect of ietS on the host cell by forming a protein complex. When a plasmid loses its toxin/antitoxin construct, ietA levels become depleted due to rapid degradation by proteases. This enables ietS to exert its toxicity on the host cell, inhibiting growth. Since cells that lose the plasmid containing the toxin/antitoxin system are terminated, these constructs are useful in selecting for a certain plasmid to be maintained.


ietAS was shown to enhance the incompatibility and stability of the Ti plasmid in Agrobacterium tumefaciens, however, the presence of ietAS showed no effect when tested in E. coli. (Yamamoto et al., 2009)

<img style="padding-top:5%" width="50%" src="IetAS.png">

Figure 1: B) Effect of transconjugation of an incoming plasmid in Agrobacterium with or without the ietAS construct. C) Effect on segregational plasmid stability of ietAS construct. A lower CFU ratio indicates a higher plasmid maintenance, demonstrating that ietAS increases plasmid stability.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 737
    Illegal BamHI site found at 1758
    Illegal XhoI site found at 659
    Illegal XhoI site found at 989
    Illegal XhoI site found at 3353
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2663
    Illegal NgoMIV site found at 3547
    Illegal AgeI site found at 3704
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 49
    Illegal BsaI.rc site found at 836
    Illegal BsaI.rc site found at 1838
    Illegal BsaI.rc site found at 3023
    Illegal BsaI.rc site found at 3305
    Illegal SapI.rc site found at 373


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