Difference between revisions of "Part:BBa K4800003"
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__NOTOC__
 
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<partinfo>BBa_K4800003 short</partinfo>
 
<partinfo>BBa_K4800003 short</partinfo>
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
===Usage and Biology===
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    <title>Document</title>
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<body>
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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</strong>
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        </div>    
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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 style="font-size: 160%; font-weight: bold;">1.Experimental Methods
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    <p>1.1 Construction of PCWJ-YciA-sfp-MmCAR-YahK<br>
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    <p>1.1 Construction of PCWJ-YciA-sfp-MmCAR-YahK<br>
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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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        <br>1.2 Construction of PRSFDuet-YciA-sfp-MmCAR-YahK<br>
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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 style="font-size: 160%; font-weight: bold;">Result
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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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    <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 </p >
 
<p>By HPLC data, it was seen that BL21 imported into PRSFDuet-YciA-sfp-MmCAR-Yahk catalyzed </p >
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4800003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4800003 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4800003 parameters</partinfo>
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<partinfo>BBa_K4800009 parameters</partinfo>
 
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Revision as of 16:36, 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.


Document

Build:

fig1.ptrc

1.Experimental Methods

1.1 Construction of PCWJ-YciA-sfp-MmCAR-YahK
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.
1.2 Construction of PRSFDuet-YciA-sfp-MmCAR-YahK
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.

Test:

Result

fig2.Catalytic generation of 1,5-PDO by BL21(DE3) containing different plasmids

By HPLC data, it was seen that BL21 imported into PRSFDuet-YciA-sfp-MmCAR-Yahk catalyzed

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 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]