Difference between revisions of "Part:BBa K4343118:Sequence, Features, and Subparts"

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URA gene is a commonly used screening marker in Y. lipolytica. Only strains with URA3 gene can grow in the medium without uracil. Since the Y. lipolytica strains we used were nutrition-deficient (uracil and leucine double deficient) and all plasmids we constructed (except BBa_K4343069) contained URA3gene, it could be verified whether the genome have been successfully edited.  
 
URA gene is a commonly used screening marker in Y. lipolytica. Only strains with URA3 gene can grow in the medium without uracil. Since the Y. lipolytica strains we used were nutrition-deficient (uracil and leucine double deficient) and all plasmids we constructed (except BBa_K4343069) contained URA3gene, it could be verified whether the genome have been successfully edited.  
 
HisG tag were utilized in combination with URA3 gene to recycle this screening marker for further use in the same strain. To be more specific, 5-fluoroorotic acid (5-FOA) could be converted into a toxic form (e.g., 5-fluorouracil) by functional OMP decarboxylase encoded by URA3 gene, thus making yeast cells unable to grow. To survive the toxicity, yeast would miss the URA3 gene by HigG tag through negative screening. In this circumstance, URA deficient strain allows for the next transformation.
 
HisG tag were utilized in combination with URA3 gene to recycle this screening marker for further use in the same strain. To be more specific, 5-fluoroorotic acid (5-FOA) could be converted into a toxic form (e.g., 5-fluorouracil) by functional OMP decarboxylase encoded by URA3 gene, thus making yeast cells unable to grow. To survive the toxicity, yeast would miss the URA3 gene by HigG tag through negative screening. In this circumstance, URA deficient strain allows for the next transformation.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4343061 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4343061 parameters</partinfo>
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Latest revision as of 05:18, 9 October 2022


hisG-URA-hisG

URA gene is a commonly used screening marker in Y. lipolytica. Only strains with URA3 gene can grow in the medium without uracil. Since the Y. lipolytica strains we used were nutrition-deficient (uracil and leucine double deficient) and all plasmids we constructed (except BBa_K4343069) contained URA3gene, it could be verified whether the genome have been successfully edited. HisG tag were utilized in combination with URA3 gene to recycle this screening marker for further use in the same strain. To be more specific, 5-fluoroorotic acid (5-FOA) could be converted into a toxic form (e.g., 5-fluorouracil) by functional OMP decarboxylase encoded by URA3 gene, thus making yeast cells unable to grow. To survive the toxicity, yeast would miss the URA3 gene by HigG tag through negative screening. In this circumstance, URA deficient strain allows for the next transformation.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 633
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 1517