Difference between revisions of "Part:BBa K3402059"

 
 
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<partinfo>BBa_K3402059 short</partinfo>
 
<partinfo>BBa_K3402059 short</partinfo>
  
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This device is composed of upSBLE (BBa_K3402030), Ptef1 (BBa_K3402007), Cas9 (BBa_K3402023), NLS (BBa_K3402008), Tsyn7 (BBa_K3402001) and doSBLE (BBa_K3402031).
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This device is composed of up<i>SBLE</i> ([https://parts.igem.org/Part:BBa_K3402030# BBa_K3402030]), P<i>tef1</i> ([https://parts.igem.org/Part:BBa_K3402007# BBa_K3402007]), <i>Cas9</i> ([https://parts.igem.org/Part:BBa_K3402023# BBa_K3402023]), <i>NLS</i> ([https://parts.igem.org/Part:BBa_K3402008# BBa_K3402008]), T<i>syn7</i> ([https://parts.igem.org/Part:BBa_K3402001# BBa_K3402001]) and do<i>SBLE</i> ([https://parts.igem.org/Part:BBa_K3402031# BBa_K3402031]).
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[[Image:Cas9 expression cassette.png|500px]]
  
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===Usage and Biology===
 
===Usage and Biology===
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The homologous arms were designed for integrating the <i>Cas9</i> gene into the genome of <i>S. Bombicola</i>. The use of the strongest promoter P<i>tef1</i> to express Cas9 protein and sgRNA could increase the gene-editing efficiency. As the Cas9 protein was too big to transport into the nucleus, a nuclear localization sequence (NLS) was needed to help the transportation of Cas9 protein.
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<br>
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The Cas9 expression cassette was fused to the self-excising hygromycin marker cassette to obtain the recombinant vector. After linearization, it was electroporated into wild-type <i>Starmerella bombicola</i>. The positive transformants were induced by galactose to eject hygromycin resistance gene. Then the recombinant strain with <i>Cas9</i> gene and hygromycin resistance gene deletion was carried on the verification experiment of single, double and triple gene-editing efficiency.
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We have built the Single-gene editing cassette ([https://parts.igem.org/Part:BBa_K3402056# BBa_K3402056]), Double-gene editing cassette ([https://parts.igem.org/Part:BBa_K3402057# BBa_K3402057]) and Triple-gene editing cassette ([https://parts.igem.org/Part:BBa_K3402058# BBa_K3402058]).
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The single gene-editing efficiency is 100%. The double gene-editing efficiency is 99%. The triple gene-editing efficiency is 30%.
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Latest revision as of 15:01, 27 October 2020


Cas9 expression cassette

This device is composed of upSBLE (BBa_K3402030), Ptef1 (BBa_K3402007), Cas9 (BBa_K3402023), NLS (BBa_K3402008), Tsyn7 (BBa_K3402001) and doSBLE (BBa_K3402031).

Cas9 expression cassette.png

Usage and Biology

The homologous arms were designed for integrating the Cas9 gene into the genome of S. Bombicola. The use of the strongest promoter Ptef1 to express Cas9 protein and sgRNA could increase the gene-editing efficiency. As the Cas9 protein was too big to transport into the nucleus, a nuclear localization sequence (NLS) was needed to help the transportation of Cas9 protein.
The Cas9 expression cassette was fused to the self-excising hygromycin marker cassette to obtain the recombinant vector. After linearization, it was electroporated into wild-type Starmerella bombicola. The positive transformants were induced by galactose to eject hygromycin resistance gene. Then the recombinant strain with Cas9 gene and hygromycin resistance gene deletion was carried on the verification experiment of single, double and triple gene-editing efficiency.


We have built the Single-gene editing cassette (BBa_K3402056), Double-gene editing cassette (BBa_K3402057) and Triple-gene editing cassette (BBa_K3402058).
The single gene-editing efficiency is 100%. The double gene-editing efficiency is 99%. The triple gene-editing efficiency is 30%.


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 2078
    Illegal BglII site found at 5642
    Illegal XhoI site found at 1399
    Illegal XhoI site found at 2128
    Illegal XhoI site found at 2491
    Illegal XhoI site found at 5443
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2905
    Illegal NgoMIV site found at 4009
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1849
    Illegal BsaI site found at 2566