Difference between revisions of "Part:BBa K4800003"
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    <title>Document</title>
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    <title>Document</title>
 
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    <p style="font-size: 180%; font-weight: bold;">Build:
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    <p style="font-size: 180%; font-weight: bold;">Build:
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        <p>< img src="https://static.igem.wiki/teams/4800/wiki/parts/ptrc-ycia-sfp-mmcar-yahk-png.png" width="80%" height="80%"></p >
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    <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/ptrc-ycia-sfp-mmcar-yahk-png.png" width="80%" height="80%"></p>
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        <div align="center">
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            <strong>fig1.ptrc</strong>
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        <strong>fig1.ptrc-ycia-sfp-mmcar-yahk</strong>
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        </div>    
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    <p style="font-size: 160%; font-weight: bold;">1.Experimental Methods
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    <p>1.Experimental methods
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    <p>1.1 Construction of PCWJ-YciA-sfp-MmCAR-YahK<br>
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1.1Construction of PCWJ-YciA-sfp-MmCAR-YahK
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        The pCWJ plasmid was extracted from a laboratory conserved strain. In-Fusion cloning was used to ligate the above vector to the Ptrc-YciA-sfp-MmCAR-YahK fragment.
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The pCWJ plasmid was extracted from a laboratory conserved strain. In-Fusion cloning was used to ligate the above vector to the Ptrc-YciA-sfp-MmCAR-YahK fragment.
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        <br>1.2 Construction of PRSFDuet-YciA-sfp-MmCAR-YahK<br>
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1.2Construction of PRSFDuet-YciA-sfp-MmCAR-YahK
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        The PRSFDuet plasmid was extracted from laboratory conserved strains. The fragment Ptrc-YciA-sfp-MmCAR-sfp-Yahk was amplified by PCR using PCWJ-YciA-sfp-MmCAR-YahK as a template, and Ptrc-YciA-MmCAR-sfp-Yahk was ligated by In-fusion cloning to the vector PRSFDuet that was linearized using PCR for homologous recombination ligation.
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The PRSFDuet plasmid was extracted from laboratory conserved strains. The fragment Ptrc-YciA-sfp-MmCAR-sfp-Yahk was amplified by PCR using PCWJ-YciA-sfp-MmCAR-YahK as a template, and Ptrc-YciA-MmCAR-sfp-Yahk was ligated by In-fusion cloning to the vector PRSFDuet that was linearized using PCR for homologous recombination ligation.
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<p style="font-size: 180%; font-weight: bold;">Test:
 
<p style="font-size: 180%; font-weight: bold;">Test:
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    <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/catalytic-generation-of-1-5-pdo-by-bl21-de3-containing-different-plasmids-png.png" width="80%" height="80%"></p>
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    <p style="font-size: 160%; font-weight: bold;">Result
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    <p>< img src="https://static.igem.wiki/teams/4800/wiki/parts/catalytic-generation-of-1-5-pdo-by-bl21-de3-containing-different-plasmids-png.png" width="80%" height="80%"></p >
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        <strong>fig2.Catalytic generation of 1,5-PDO by BL21(DE3) containing different plasmids</strong>
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         <strong>fig2.Catalytic generation of 1,5-PDO by BL21(DE3) containing different plasmids</strong>
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<p>By HPLC data, it was seen that BL21 imported into PRSFDuet-YciA-sfp-MmCAR-Yahk catalyzed the production of more 1,5-PDO.</p >
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    </div>
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    <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/fermentation-of-ka30-containing-different-plasmids-to-produce-1-5-pdo-png.png" width="80%" height="80%"></p>
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<p>By HPLC data, it was seen that BL21 imported into PRSFDuet-YciA-sfp-MmCAR-Yahk catalyzed </p >
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        <strong>fig3.Fermentation of KA30 containing different plasmids to produce 1,5-PDO</strong>
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<p>By comparing the fermentation production of 1,5-PDO content with the previous KA30 with PCWJ+PTRC99a, we can clearly see that KA30 with PRSFDuet+PTRC99a produced more 1,5-PDO, therefore, we decided to use both vectors, PRSFDuet and PTRC99a, in all our future experiments.</p >
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Revision as of 16:29, 6 October 2023


Ptrc-sfp-MmCAR-YahK

(1)Ptrc is a promoter.(2)YahK is a acyl-CoA thioester hydrolase from Haemophilus influenzae.(3) Sfp is a phosphopantetheinyl transferase from Bacilus subtilis.(4) MmCAR is a Carboxylic acid reductase from Mycobacterium marinum.(5) YahK is a NADPH-dependent aldehyde reductase.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1332
    Illegal PstI site found at 1767
    Illegal PstI site found at 1794
    Illegal PstI site found at 3063
    Illegal PstI site found at 3090
    Illegal PstI site found at 3483
    Illegal PstI site found at 3979
    Illegal PstI site found at 4623
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1332
    Illegal NheI site found at 4891
    Illegal PstI site found at 1767
    Illegal PstI site found at 1794
    Illegal PstI site found at 3063
    Illegal PstI site found at 3090
    Illegal PstI site found at 3483
    Illegal PstI site found at 3979
    Illegal PstI site found at 4623
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1332
    Illegal BglII site found at 2321
    Illegal BglII site found at 2947
    Illegal BglII site found at 3223
    Illegal BglII site found at 3914
    Illegal BamHI site found at 1304
    Illegal XhoI site found at 4863
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1332
    Illegal PstI site found at 1767
    Illegal PstI site found at 1794
    Illegal PstI site found at 3063
    Illegal PstI site found at 3090
    Illegal PstI site found at 3483
    Illegal PstI site found at 3979
    Illegal PstI site found at 4623
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1332
    Illegal PstI site found at 1767
    Illegal PstI site found at 1794
    Illegal PstI site found at 3063
    Illegal PstI site found at 3090
    Illegal PstI site found at 3483
    Illegal PstI site found at 3979
    Illegal PstI site found at 4623
    Illegal NgoMIV site found at 3591
    Illegal NgoMIV site found at 3653
    Illegal NgoMIV site found at 3870
    Illegal NgoMIV site found at 4317
    Illegal AgeI site found at 1497
    Illegal AgeI site found at 2508
    Illegal AgeI site found at 2805
    Illegal AgeI site found at 3690
    Illegal AgeI site found at 3699
    Illegal AgeI site found at 4374
  • 1000
    COMPATIBLE WITH RFC[1000]