Difference between revisions of "Part:BBa I742151"
 
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===Usage and Biology===
 
===Usage and Biology===
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[https://2022.igem.wiki/fudan/  Team Fudan 2022]
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[https://2022.igem.wiki/fudan/  Team Fudan 2022] used BBa I742151 in retinoid biosynthesis in E. coli.
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used BBa I742151 in retinoid biosynthesis by E. coli.
  
 
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<partinfo>BBa_I742151 parameters</partinfo>
 
<partinfo>BBa_I742151 parameters</partinfo>
 
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<h2>Contribution by Team 2024 Foshan-GreatBay</h2>
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  <h3>Summary</h3>
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    <p>
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        To increase the yield of β-carotene in yeast cells, we constructed a new composite part BBa_K5419000 (pX-2-PaCrtE) with crtE (BBa_I742151) gene fragment. This composited part was used together with other composite parts, BBa_K5419005 (pXII-5-BtCrtI), and BBa_K5419009 (pXI-2-XdCrtYB), for the construction of yeast strains with high β-carotene production.
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    <h3>Construction Design</h3>
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        We constructed the plasmid by placing the gene under the regulation of a strong constitutive GAP promoter and a CYC terminator, respectively. Integration sequence was added upstream and downstream of the expression cassette to integrate the target gene into the genome of <i>S. cerevisiae</i> using the CRISPR/Cas9 system (Figure 1).
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    </p>
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    <!-- Figure 1 -->
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    <div style="text-align:center;">
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        "https://static.igem.wiki/teams/5419/bba-k5419000/figure-1.jpg"
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        <div class="caption">Figure 1 Design diagrams of pX-2-PaCrtE.</div>
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    </div>
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    <h3>Experimental Approach</h3>
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    <h4>Construction of integration plasmids</h4>
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    <p>
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        Firstly, we obtained the target gene expression frames (GAP promotor-gene-CYC terminator) by PCR amplification, and agarose gel electrophoresis results showed that we succeeded in obtaining these fragments. Next, we double-digested the target fragment and the vector (containing the <i>S. cerevisiae</i> X-2 integration site genes) and obtained the plasmid by enzymatic ligation. Finally, we transformed the enzyme-ligation product into <i>E. coli</i> DH5α competent cells, and the colony PCR and sequencing results showed that we successfully constructed the plasmid (Figure 2).
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    </p>
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    <!-- Figure 2 -->
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    <div style="text-align:center;">
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        "https://static.igem.wiki/teams/5419/bba-k5419000/figure-2.jpg"
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        <div class="caption">Figure 2 The construction results of the pX-2-PaCrtE plasmid.</div>
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    </div>

Latest revision as of 04:19, 2 October 2024


crtE (geranylgeranyl pyrophosphate synthase) coding sequence.

crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis parthway.


Usage and Biology

Team Fudan 2022 used BBa I742151 in retinoid biosynthesis by E. coli.

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 AgeI site found at 715
  • 1000
    COMPATIBLE WITH RFC[1000]



Contribution by Team 2024 Foshan-GreatBay

Summary

To increase the yield of β-carotene in yeast cells, we constructed a new composite part BBa_K5419000 (pX-2-PaCrtE) with crtE (BBa_I742151) gene fragment. This composited part was used together with other composite parts, BBa_K5419005 (pXII-5-BtCrtI), and BBa_K5419009 (pXI-2-XdCrtYB), for the construction of yeast strains with high β-carotene production.

Construction Design

We constructed the plasmid by placing the gene under the regulation of a strong constitutive GAP promoter and a CYC terminator, respectively. Integration sequence was added upstream and downstream of the expression cassette to integrate the target gene into the genome of S. cerevisiae using the CRISPR/Cas9 system (Figure 1). 

       "figure-1.jpg"
Figure 1 Design diagrams of pX-2-PaCrtE.

Experimental Approach

Construction of integration plasmids

Firstly, we obtained the target gene expression frames (GAP promotor-gene-CYC terminator) by PCR amplification, and agarose gel electrophoresis results showed that we succeeded in obtaining these fragments. Next, we double-digested the target fragment and the vector (containing the S. cerevisiae X-2 integration site genes) and obtained the plasmid by enzymatic ligation. Finally, we transformed the enzyme-ligation product into E. coli DH5α competent cells, and the colony PCR and sequencing results showed that we successfully constructed the plasmid (Figure 2). 

       "figure-2.jpg"
Figure 2 The construction results of the pX-2-PaCrtE plasmid.