Difference between revisions of "Part:BBa K2179003"
 
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<h5>Gene Design</h5>
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<h5>Translational Unit for Chlorite Dismutase Cld(-SP)</h5>
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<p>Chlorite Dismutase is the second enzyme utilized in a number of bacterial species' perchlorate reducing pathway. In their original work, Thorell et al. cloned, sequenced, and functionally expressed the natural coding sequence for Chlorite Dismutase from <i>I. dechloratans.</i> They determined that the gene was capable of converting ClO<sub>2</sub><sup>-</sup> ions to O<sub>2</sub> gas and Cl<sup>-</sup> ions and that its activity was localized to the periplasm, even though its putative signal peptide had not been cleaved. We designed this Cld(-SP) gene to lack this putative signal sequence. The purpose of this modification was so that once translation was complete, the Chlorite Dismutase enzyme would remain in the cytoplasm of our <i>E. coli.</i> chassis. A C-terminal histidine tag was also to this translational unit in order to purify the enzymes in the event that O<sub>2</sub> production from living cells proved inefficient. Flanking these BsaI sites were XbaI (5’) and the BBa_ std 1 suffix (3’) to facilitate parts creation for the iGEM Registry. We note that the natural coding sequence contained none of the standard BBa_10 restrictions sites, but did contain a single internal BsaI site which we eliminated with a single base silent substitution.
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Chlorite Dismutase is an enzyme which catalyzes the reduction of chlorite ions to oxygen gas and chloride ions. Cld(-SP) is a translational unit which lacks Chlorite Dismutase original putative signal peptide, and also has a polyhistidine tag appended on it for the purpose of protein purification. The activity of this enzyme can be observed as bubbling of the liquid culture when transformed cells are combined with compounds containing the chlorite oxyanion.  
 
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<h5>Functionality</h5>
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<partinfo>BBa_K2179003 SequenceAndFeatures</partinfo>
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<p>An oxygen production assay was conducted where transformed <i>E. coli</i> was combined with solid Sodium Chlorite. Almost immediately, the formation of a gas, which was inferred to be oxygen, was noted. This indicates the proper functioning of our synthetic G-Block and aligns with the results of Thorell et. al.
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<h5>References</h5>
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<p>
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Thorell, H.D, Karlsson, J., Portelius, E.and Nilsson, T. Biochimica et Biophysica Acta 1577 (2002) 445–451
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Samant,S., Gupta, G., Karthikeyan, S., Haq, S., Sambasivam, N.G., and Sukumaran, S. J Ind Microbiol Biotechnol (2014) 41:1435–1442
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</p>
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Latest revision as of 23:48, 29 October 2016

Translational Unit for Chlorite Dismutase Cld(-SP)

Chlorite Dismutase is an enzyme which catalyzes the reduction of chlorite ions to oxygen gas and chloride ions. Cld(-SP) is a translational unit which lacks Chlorite Dismutase original putative signal peptide, and also has a polyhistidine tag appended on it for the purpose of protein purification. The activity of this enzyme can be observed as bubbling of the liquid culture when transformed cells are combined with compounds containing the chlorite oxyanion.

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 807
    Illegal BsaI.rc site found at 6